Methods for degrading lignocellulosic materials

ABSTRACT

The present invention relates to methods for degrading a lignocellulosic material, comprising: treating the lignocellulosic material with an effective amount of one or more cellulolytic enzymes in the presence of at least one surfactant selected from the group consisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein the presence of the surfactant increases the degradation of lignocellulosic material compared to the absence of the surfactant. The present invention also relates to methods for producing an organic substance, comprising: (a) saccharifying a lignocellulosic material with an effective amount of one or more cellulolytic enzymes in the presence of at least one surfactant selected from the group consisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein the presence of the surfactant increases the degradation of lignocellulosic material compared to the absence of the surfactant; (b) fermenting the saccharified lignocellulosic material of step (a) with one or more fermentating microoganisms; and (c) recovering the organic substance from the fermentation.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/537,452, filed Jan. 16, 2004, which application is incorporatedherein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under NREL SubcontractNo. ZCO-30017-02, Prime Contract DE-AC36-98GO10337 awarded by theDepartment of Energy. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for degrading lignocellulosicmaterials.

2. Description of the Related Art

The majority of carbohydrates in plants are in the form oflignocellulose, which is composed of mainly cellulose, hemicellulose,pectin, and lignin. Lignocellulose is found, for example, in the stems,leaves, hulls, husks, and cobs of plants. Hydrolysis of these polymersreleases a mixture of neutral sugars including glucose, xylose, mannose,galactose, and arabinose.

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, glucohydrolases and beta-glucosidases.Endoglucanases digest the cellulose polymer at random locations, openingit to attack by cellobiohydrolases. Glucohydrolases liberate moleculesof glucose from the ends of the cellulose polymer. Cellobiohydrolasessequentially release molecules of cellobiose from the ends of thecellulose polymer. Cellobiose is a water-soluble beta-1,4-linked dimerof glucose. Beta-glucosidases hydrolyze cellobiose to glucose.

Hemicelluloses are short branched chain heteropolysaccharides that arecomposed of various hexoses (glucose, mannose and galactose), pentoses(D-xylose and L-arabinose), uronic acids, acetic acid, and other minorsugars. Similar to cellulose degradation, hemicellulose hydrolysisrequires coordinated action of many enzymes, which can be placed intothree general categories, the endo-acting enzymes that attack internalbonds within the polysaccharide chain, the exo-acting enzymes that actprocessively from either the reducing or nonreducing end of thepolysaccharide chain, and the accessory enzymes (acetylesterases andesterases that hydrolyze lignin glycoside bonds).

Lignocellulosic materials, such as wood, herbaceous material,agricultural residues, corn fiber, waste paper, pulp and paper millresidues can be used to produce ethanol. No known natural organism canrapidly and efficiently metabolize all carbohydrate polymers inlignocellulosic biomass into ethanol. The conversion of lignocellulosicfeedstocks into ethanol has the advantages of the ready availability oflarge amounts of feedstock, the desirability of avoiding burning or landfilling the materials, and the cleanliness of the ethanol fuel. Once thecellulose is converted to glucose, the glucose is easily fermented byyeast into ethanol.

However, an obstacle to commercialization is the cost of enzymes toconvert the lignocellulosic material to glucose and other fermentablesugars. There is a need in the art to improve the ability ofcellulolytic enzymes to degrade lignocellulosic materials to usefulorganic products or to intermediates to useful end-products.

It is an object of the present invention to improve the ability ofcellulolytic enzymes to degrade lignocellulosic materials.

SUMMARY OF THE INVENTION

The present invention relates to methods for degrading a lignocellulosicmaterial, comprising: treating the lignocellulosic material with aneffective amount of one or more cellulolytic enzymes in the presence ofat least one surfactant selected from the group consisting of asecondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenolethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein thepresence of the surfactant increases the degradation of lignocellulosicmaterial compared to the absence of the surfactant.

The present invention also relates to methods for producing an organicsubstance, comprising:

-   -   (a) saccharifying a lignocellulosic material with an effective        amount of one or more cellulolytic enzymes in the presence of at        least one surfactant selected from the group consisting of a        secondary alcohol ethoxylate, fatty alcohol ethoxylate,        nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene        ether, wherein the presence of the surfactant increases the        degradation of lignocellulosic material compared to the absence        of the surfactant;    -   (b) fermenting the saccharified lignocellulosic material of        step (a) with one or more fermenting microoganisms; and    -   (c) recovering the organic substance from the fermentation.

In a preferred embodiment, the organic substance is alcohol.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pAILo1.

FIG. 2 shows a restriction map of pMJ04.

FIG. 3 shows a restriction map of pCaHj527.

FIG. 4 shows a restriction map of pMT2188.

FIG. 5 shows a restriction map of pCaHj568.

FIG. 6 shows a restriction map of pMJ05.

FIG. 7 shows the DNA sequence (SEQ ID NO: 26) and deduced amino acidsequence (SEQ ID NO: 27) of the secretion signal sequence of anAspergillus oryzae beta-glucosidase.

FIG. 8 shows the DNA sequence (SEQ ID NO: 30) and deduced amino acidsequence (SEQ ID NO: 31) of the secretion signal sequence of a Humicolainsolens endoglucanase V.

FIG. 9 shows a restriction map of pSMail35.

FIGS. 10A and 10B shows the genomic DNA sequence and the deduced aminoacid sequence of an Aspergillus fumigatus beta-glucosidase (SEQ ID NOS:36 and 37, respectively). The predicted signal peptide is underlined andpredicted introns are italicized.

FIG. 11 shows a restriction map of pBANe10.

FIG. 12 shows a restriction map of pAlLo2.

FIG. 13 shows a restriction map of pEJG97.

FIG. 14 shows a restriction map of pMJ06.

FIG. 15 shows a restriction map of pMJ09.

FIG. 16 shows a restriction map of pJZHEG1.

FIG. 17 shows the effect of Softanol® 90 (0.1 ml/g PCS) on hydrolysis ofPCS (5%) by Celluclast 1.5 L at 50° C.

FIG. 18 shows the effect of Lutensol® AT80 (0.1 ml/g PCS) on hydrolysisof PCS (5%) by Celluclast 1.5 L at 50° C.

FIG. 19 shows the effect of Softanol® 90 (0.2 ml/g PCS) on hydrolysis ofPCS (5%) by Celluclast 1.5 L at 50° C.

FIG. 20 shows the effect of Softanol® 90 (0.2 ml/g PCS) on hydrolysis ofPCS (5%) by Celluclast 1.5 L at 55° C.

FIG. 21 shows the Softanol® 90 dose dependence for hydrolysis of PCS(5%) by Celluclast 1.5 L (2 mg/g PCS) at 50° C.

FIG. 22 shows the effect of different Softanol® products on hydrolysisof PCS (5%) by Celluclast 1.5 L (2 mg/g PCS) at 50° C.

FIG. 23 shows the effect of Softanol□ 90 (0.2 ml/g PCS) on hydrolysis ofPCS (5%) by Celluclast 1.5 L (2 mg/g PCS) supplemented with Aspergillusoryzae beta-glucosidase (0.06 mg/g PCS) and cell-free broth ofTrichoderma reesei expressing Aspergillus oryzae beta-glucosidase (2mg/g PCS) at 50° C.

FIG. 24 shows the effect of Softanol® 90 (0.1 ml/g PCS) on 24 hourconversion of ethanol washed/milled PCS (1%) by purified Trichodermareesei cellobiohydrolase 1 (2-10 mg/g PCS) at 40-65° C.

FIG. 25 shows the effect of Softanol® 90 (0.1 ml/g PCS) on 24 hourconversion of ethanol washed/milled PCS (1%) by purified Trichodermareesei cellobiohydrolase 1 (2-10 mg/g PCS) supplemented with 5%Aspergillus fumigatus beta-glucosidase (0.1-0.5 mg/g PCS) at 40-65° C.

FIG. 26 shows the effect of Softanol® 90 (0.1 ml/g PCS) on 24 hourconversion of ethanol washed/milled PCS (1%) by purified Trichodermareesei cellobiohydrolase 1 (2-20 mg/g PCS) supplemented with 20%Trichoderma reesei endoglucanase I (0.4-4 mg/g PCS) and 5% Aspergillusfumigatus beta-glucosidase (0.1-1 mg/g PCS) at 40-65° C.

FIG. 27 shows the effect of Softanol® 90 (0.1 ml/g PCS) on 24 hourconversion of ethanol washed/milled PCS (1%) by purified Trichodermareesei cellobiohydrolase 1 (2-20 mg/g PCS) supplemented with 20%Acidothermus cellulolyticus E1cd (0.4-4 mg/g PCS) and 5% Aspergillusfumigatus beta-glucosidase (0.1-1 mg/g PCS) at 40-65° C.

FIG. 28 shows the effect of Softanol® (0.05-0.2 ml/g cellulose) onhydrolysis of Avicel (1%) by Celluclast 1.5 L (1.25-20 mg/cellulose) at50° C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for degrading a lignocellulosicmaterial, comprising: treating the lignocellulosic material with aneffective amount of one or more cellulolytic enzymes in the presence ofat least one surfactant selected from the group consisting of asecondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenolethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein thepresence of the surfactant increases the degradation of lignocellulosicmaterial compared to the absence of the surfactant.

Lignocellulosic Material

In the methods of the present invention, the lignocellulosic materialcan be any material containing lignocellulose. Lignocellulose isgenerally found, for example, in the stems, leaves, hulls, husks, andcobs of plants or leaves, branches, and wood of trees. Thelignocellulosic material can also be, but is not limited to, herbaceousmaterial, agricultural residues, forestry residues, municipal solidwastes, waste paper, and pulp and paper mill residues.

In a preferred embodiment, the lignocellulosic material is corn stover.In another preferred embodiment, the lignocellulosic material is cornfiber. In another preferred embodiment, the lignocellulosic material isrice straw. In another preferred embodiment, the lignocellulosicmaterial is paper and pulp processing waste. In another preferredembodiment, the lignocellulosic material is woody or herbaceous plants.

The lignocellulosic material may be used as is or may be subjected to apretreatment using conventional methods known in the art. For example,physical pretreatment techniques can include various types of milling,irradiation, steaming/steam explosion, and hydrothermolysis; chemicalpretreatment techniques can include dilute acid, alkaline, organicsolvent, ammonia, sulfur dioxide, carbon dioxide, and pH-controlledhydrothermolysis; and biological pretreatment techniques can involveapplying lignin-solubilizing microorganisms (see, for example, Hsu,T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh, P., Singh, A., 1993, Physicochemicaland biological treatments for enzymatic/microbial conversion oflignocellulosic biomass, Adv. Appl. Microbiol., 39: 295-333; McMillan,J. D., 1994, Pretreating lignocellulosic biomass: a review, in EnzymaticConversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O.,and Overend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson, L., andHahn-Hagerdal, B., 1996, Fermentation of lignocellulosic hydrolysatesfor ethanol production, Enz. Microb. Tech, 18: 312-331; and Vallander,L., and Eriksson, K.-E. L., 1990, Production of ethanol fromlignocellulosic materials: State of the art, Adv. Biochem.Eng./Biotechnol., 42: 63-95).

Surfactants

In the methods of the present invention, the surfactant may be anysurfactant selected from the group consisting of a secondary alcoholethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecylethoxylate, and polyoxyethylene ether. The surfactant is addedexogenously.

In a preferred embodiment, the surfactant is a secondary alcoholethoxylate.

In another preferred embodiment, the surfactant is a fatty alcoholethoxylate.

In another preferred embodiment, the surfactant is a nonylphenolethoxylate.

In another preferred embodiment, the surfactant is a tridecylethoxylate.

In another preferred embodiment, the surfactant is a polyoxyethyleneether.

A secondary alcohol ethoxylate surfactant has the formulaC₁₁₋₁₅H₂₃₋₃₁O(CH₂CH₂O)_(x)H, wherein x is the degree of ethoxylation.The degree of ethoxylation can be from at least 3 to at least 50. In apreferred embodiment, the secondary alcohol ethoxylate isalkyloxypolyethyleneoxyethanol 50 (x=5). In another preferredembodiment, the secondary alcohol ethoxylate isalkyloxypolyethyleneoxyethanol 90 (x=9). In another preferredembodiment, the secondary alcohol ethoxylate isalkyloxypolyethyleneoxyethanol 120 (x=12). In another preferredembodiment, the secondary alcohol ethoxylate isalkyloxypolyethyleneoxyethanol 200 (x=20). Examples of commerciallyavailable secondary alcohol ethoxylate surfactants include, but are notlimited to, SOFTANOL™ 50, SOFTANOL™ 90, SOFTANOL™ 120, and SOFTANOL™200, obtainable from INEOS Oxide, Zwijndrecht, Belgium.

A fatty alcohol ethoxylate has the formula C₁₆₋₁₈H₃₃₋₃₇O(CH₂CH₂O)_(x)H,wherein x is the degree of ethoxylation. The degree of ethoxylation canbe from at least 11 to at least 80. Examples of commercially availablefatty alcohol ethoxylate surfactants include, but are not limited to,Lutensol AT50 (x=50) and Lutensol AT80 (x=80), obtainable from BASFCorp., Mount Olive, N.J., USA.

A nonylphenol ethoxylate has the formula C₉H₁₉C₆H₄(CH₂CH₂O)_(x)OH,wherein x is the degree of ethoxylation. The degree of ethoxylation canbe from at least 4 to at least 70. Examples of commercially availablenonylphenol ethoxylate surfactants include, but are not limited to,Tergitol NP-9 (x=9.3), obtainable from the Dow Chemical Company,Midland, Mich., USA.

A tridecyl ethoxylate has the formula C₁₃₋₁₅H₂₇₋₃₁O(CH₂CH₂O)_(x)H,wherein x is the degree of ethoxylation. The degree of ethoxylation canbe from at least 2 to at least 50. Examples of commercially availabletridecyl ethoxylate surfactants include, but are not limited to, NovellII TDA-6.6 (x=6.6) and Novell II TDA-8.5 (x=8.5), obtainable from Sasol,Houston, Tex.

A polyoxyethylene ether has the formulaC₁₂₋₁₈H₂₅₋₃₇(CH₂CH₂O)_(x)OCH₂CHO, wherein x is the degree ofethoxylation. The degree of ethoxylation can be from at least 10 to atleast 23. Examples of commercially available polyoxyethylene ethersurfactants include, but are not limited to, Brij 35 (x=23), Brij 56(x=10), Brij 97 (x=10), and Brij 98 (x=20), obtainable from SigmaChemical Co., St. Loius, Mo.

A summary of the above-noted surfactants is shown in Table 1. TABLE 1CP, EO Surfactant Chemistry Supplier MW ° C. HLB Mole # AppearanceSoftanol 50 Secondary AE Honeywell & 420 <0 10.5 5 LiquidC₁₁₋₁₅H₂₃₋₃₁O(EO)₅H Stein/INEO S Oxide Sofftanol 90 Secondary AEHoneywell & 600 56 13.3 9 Liquid C₁₁₋₁₅H₂₃₋₃₁O(EO)₉H Stein/INEO S OxideSoftanol 120 Secondary AE Honeywell & 730 83 14.5 12 WaxC₁₁₋₁₅H₂₃₋₃₁O(EO)₁₂H Stein/INEO (Liquid @ S Oxide 30° C.) Softanol 200Secondary AE Honeywell & 1080  NA 20 Solid C₁₁₋₁₅H₂₃₋₃₁O(EO)₂₀HStein/INEO S Oxide Lutensol AT50 Fatty AE BASF 92 50 SolidC₁₆₋₁₈H₃₃₋₃₇O(EO)₅₀H Lutensol AT80 Fatty AE BASF 80 SolidC₁₆₋₁₈H₃₃₋₃₇O(EO)₈₀H Tergitol NP-9 Nonylphenol Dow 53 13 9.3 LiquidEthoxylate Chemical C₉H₁₉C₆H₄(EO)_(9.3)OH Company Novell II TDA-Tridecyl AE Sasol North 25 11.8 6.7 Liquid 6.6 C₁₃₋₁₅H₂₇₋₃₁O(EO)_(6.6)HAmerica Novell II TDA- Tridecyl AE Sasol North 54 13 8.7 Liquid 8.5C₁₃₋₁₅H₂₇₋₃₁O(EO)_(8.5)H America Brij 35 Polyoxyethylene 23 ICI LiquidLauryl Ether Americas/ Sigma Brij 56 Polyoxyethylene 10 ICI Wax CetylEther Americas/ Sigma Brij 97 Polyoxyethylene 10 ICI Wax Oleyl EtherAmericas/ Sigma Brij 98 Polyoxyethylene 20 ICI Wax Oleyl Ether Americas/SigmaAE—alcohol ethoxylate,EO—ethylene oxide (CH₂CH₂O),PO—propylene oxide (CH₂CHCH₃O),CP—cloud point,HLB—hydrophilic/lipophilic balanceCellulolytic Enzymes

In the methods of the present invention, the cellulolytic enzyme may anyenzyme involved in the degradation of lignocellulose to glucose, xylose,mannose, galactose, and arabinose. The cellulolytic enzyme may be amulticomponent enzyme preparation, e.g., cellulase, a monocomponentenzyme preparation, e.g., endoglucanase, cellobiohydrolase,glucohydrolase, beta-glucosidase, or a combination of multicomponent andmonocomponent enzymes. The cellulolytic enzymes may have activity, i.e.,hydrolyze cellulose, either in the acid, neutral, or alkaline pH-range.

The cellulolytic enzyme may be of fungal or bacterial origin, which maybe obtainable or isolated and purified from microorganisms which areknown to be capable of producing cellulolytic enzymes, e.g., species ofHumicola, Coprinus, Thielavia, Fusarium, Myceliophthora, Acremonium,Cephalosporium, Scytalidium, Penicillium or Aspergillus (see, forexample, EP 458162), especially those produced by a strain selected fromthe species Humicola insolens (reclassified as Scytalidium thermophilum,see for example, U.S. Pat. No. 4,435,307), Coprinus cinereus, Fusariumoxysporum, Myceliophthora thermophila, Meripilus giganteus, Thielaviaterrestris, Acremonium sp., Acremonium persicinum, Acremoniumacremonium, Acremonium brachypenium, Acremonium dichromosporum,Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum,Acremonium incoloratum, and Acremonium furatum; preferably from thespecies Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672,Myceliophthora thermophila CBS 117.65, Cephalosporium sp. RYM-202,Acremonium sp. CBS 478.94, Acremonium sp. CBS 265.95, Acremoniumpersicinum CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporiumsp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremoniumdichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremoniumpinkertoniae CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremoniumincoloratum CBS 146.62, and Acremonium furatum CBS 299.70H. Cellulolyticenzymes may also be obtained from Trichoderma (particularly Trichodermaviride, Trichoderma reesei, and Trichoderma koningii), alkalophilicBacillus (see, for example, U.S. Pat. No. 3,844,890 and EP 458162), andStreptomyces (see, for example, EP 458162).

The cellulolytic enzymes used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and cellulase production areknown in the art (see, e.g., Bailey, J. E., and Ollis, D. F.,Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY,1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of a cellulolytic enzyme. Fermentation may,therefore, be understood as comprising shake flask cultivation, small-or large-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentersperformed in a suitable medium and under conditions allowing thecellulase to be expressed or isolated.

The resulting cellulolytic enzymes produced by the methods describedabove may be recovered from the fermentation medium by conventionalprocedures including, but not limited to, centrifugation, filtration,spray-drying, evaporation, or precipitation. The recovered enzyme maythen be further purified by a variety of chromatographic procedures,e.g., ion exchange chromatography, gel filtration chromatography,affinity chromatography, or the like.

Cellulase hydrolyzes carboxymethyl cellulose (CMC), thereby decreasingthe viscosity of the incubation mixture. The resulting reduction inviscosity may be determined by a vibration viscosimeter (e.g., MIVI 3000from Sofraser, France). Determination of cellulase activity, measured interms of Cellulase Viscosity Unit (CEVU), quantifies the amount ofcatalytic activity present in a sample by measuring the ability of thesample to reduce the viscosity of a solution of carboxymethyl cellulose(CMC). The assay is carried out at 40° C. in 0.1 M phosphate pH 9.0buffer for 30 minutes with CMC as substrate (33.3 g/L carboxymethylcellulose Hercules 7 LFD) and an enzyme concentration of approximately3.3-4.2 CEVU/ml. The CEVU activity is calculated relative to a declaredenzyme standard, such as Celluzyme™ Standard 17-1194 (obtained fromNovozymes A/S, Bagsvaerd, Denmark).

Examples of cellulases suitable for use in the present inventioninclude, for example, CELLUCLAST™ (available from Novozymes A/S) andNOVOZYM™ 188 (available from Novozymes A/S). Other commerciallyavailable preparations comprising cellulase which may be used includeCELLUZYME™, CEREFLO™ and ULTRAFLO™ (Novozymes A/S), LAMINEX™ andSPEZYME™ CP (Genencor Int.), and ROHAMENT™ 7069 W (Röhm GmbH). Thecellulase enzymes are added in amounts effective from about 0.001% toabout 5.0% wt. of solids, more preferably from about 0.025% to about4.0% wt. of solids, and most preferably from about 0.005% to about 2.0%wt. of solids.

As mentioned above, the cellulolytic enzymes used in the methods of thepresent invention may be monocomponent preparations, i.e., a componentessentially free of other cellulase components. The single component maybe a recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). Other examples of monocomponent cellulolytic enzymesinclude, but are not limited to, those disclosed in JP-07203960-A andWO-9206209. The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticenzymes may also be prepared by purifying such an enzyme from afermentation medium.

Examples of monocomponent cellulolytic enzymes useful in practicing themethods of the present invention include, but are not limited to,endoglucanase, cellobiohydrolase, glucohydrolase, and beta-glucosidase.

The term “endoglucanase” is defined herein as anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. No. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

The exo-1,4-beta-D-glucanases include both cellobiohydrolases andglucohydrolases.

The term “cellobiohydrolase” is defined herein as a 1,4-beta-D-glucancellobiohydrolase (E.C. 3.2.1.91), which catalyzes the hydrolysis of1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, orany beta-1,4-linked glucose containing polymer, releasing cellobiosefrom the reducing or non-reducing ends of the chain. For purposes of thepresent invention, cellobiohydrolase activity is determined according tothe procedures described by Lever et al., 1972, Anal. Biochem. 47:273-279 and by van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156;van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288. In thepresent invention, the Lever et al. method was employed to assesshydrolysis of cellulose in corn stover, while the method of vanTilbeurgh et al. was used to determine the cellobiohydrolase activity ona fluorescent disaccharide derivative.

The term “glucohydrolase” is defined herein as a 1,4-beta-D-glucanglucohydrolase (E.C. 3.2.1.74), which catalyzes the hydrolysis of1,4-linkages (O-glycosyl bonds) in 1,4-beta-D-glucans so as to removesuccessive glucose units. For purposes of the present invention,exoglucanase activity is determined according to the procedure describedby Himmel et al., 1986, J. Biol. Chem. 261: 12948-12955.

The term “beta-glucosidase” is defined herein as a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis ofterminal non-reducing beta-D-glucose residues with the release ofbeta-D-glucose. For purposes of the present invention, beta-glucosidaseactivity is determined according to the basic procedure described byVenturi et al., 2002, J. Basic Microbiol. 42: 55-66, except differentconditions were employed as described herein. One unit ofbeta-glucosidase activity is defined as 1.0 μmole of p-nitrophenolproduced per minute at 50° C., pH 5 from 4 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate, 0.01% Tween-20.

Processing of Lignocellulosic Materials

The methods of the present invention may be used to process alignocellulosic material to many useful organic products, chemicals andfuels. In addition to ethanol, some commodity and specialty chemicalsthat can be produced from lignocellulose include xylose, acetone,acetate, glycine, lysine, organic acids (e.g., lactic acid),1,3-propanediol, butanediol, glycerol, ethylene glycol, furfural,polyhydroxyalkanoates, cis, cis-muconic acid, and animal feed (Lynd, L.R., Wyman, C. E., and Gerngross, T. U., 1999, Biocommodity engineering,Biotechnol. Prog., 15: 777-793; Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; and Ryu, D. D. Y., and Mandels, M., 1980, Cellulases:biosynthesis and applications, Enz. Microb. Technol., 2: 91-102).Potential coproduction benefits extend beyond the synthesis of multipleorganic products from fermentable carbohydrate. Lignin-rich residuesremaining after biological processing can be converted to lignin-derivedchemicals, or used for power production.

Conventional methods used to process the lignocellulosic material inaccordance with the methods of the present invention are well understoodto those skilled in the art. The methods of the present invention may beimplemented using any conventional biomass processing apparatusconfigured to operate in accordance with the invention.

Such an apparatus may include a batch-stirred reactor, a continuous flowstirred reactor with ultrafiltration, a continuous plug-flow columnreactor (Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of theenzymatic hydrolysis of cellulose: 1. A mathematical model for a batchreactor process, Enz. Microb. Technol., 7: 346-352), an attritionreactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion of wastecellulose by using an attrition bioreactor, Biotechnol. Bioeng., 25:53-65), or a reactor with intensive stirring induced by anelectromagnetic field (Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y.,Davydkin, V. Y., Protas, O. V., 1996, Enhancement of enzymatic cellulosehydrolysis using a novel type of bioreactor with intensive stirringinduced by electromagnetic field, Appl. Biochem. Biotechnol., 56:141-153).

The conventional methods include, but are not limited to,saccharification, fermentation, separate hydrolysis and fermentation(SHF), simultaneous saccharification and fermentation (SSF),simultaneous saccharification and cofermentation (SSCF), hybridhydrolysis and fermentation (HHF), and direct microbial conversion(DMC).

SHF uses separate process steps to first enzymatically hydrolyzecellulose to glucose and then ferment glucose to ethanol. In SSF, theenzymatic hydrolysis of cellulose and the fermentation of glucose toethanol is combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF includes the coferementation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog., 15: 817-827).HHF includes two separate steps carried out in the same reactor but atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(cellulase production, cellulose hydrolysis, and fermentation) in onestep (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S.,2002, Microbial cellulose utilization: Fundamentals and biotechnology,Microbiol. Mol. Biol. Reviews, 66: 506-577).

“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. A fermentationprocess includes, without limitation, fermentation processes used toproduce fermentation products including alcohols (e.g., arabinitol,butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, andxylitol); organic acids (e.g., acetic acid, acetonic acid, adipic acid,ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid,fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaricacid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,malonic acid, oxalic acid, propionic acid, succinic acid, and xylonicacid); ketones (e.g., acetone); amino acids (e.g., aspartic acid,glutamic acid, glycine, lysine, serine, and threonine); gases (e.g.,methane, hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)).Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry.

Methods for Producing Organic Substances

The present invention also relates to methods for producing an organicsubstance, comprising: (a) saccharifying a lignocellulosic material withan effective amount of one or more cellulolytic enzymes in the presenceof at least one surfactant selected from the group consisting of asecondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenolethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein thepresence of the surfactant increases the degradation of lignocellulosicmaterial compared to the absence of the surfactant; (b) fermenting thesaccharified lignocellulosic material of step (a) with one or morefermentating microoganisms; and (c) recovering the organic substancefrom the fermentation.

The organic substance can be any substance derived from thefermentation. In a preferred embodiment, the organic substance is analcohol. It will be understood that the term “alcohol” encompasses anorganic substance that contains one or more hydroxyl moieties. In a morepreferred embodiment, the alcohol is arabinitol. In another morepreferred embodiment, the alcohol is butanol. In another more preferredembodiment, the alcohol is ethanol. In another more preferredembodiment, the alcohol is glycerol. In another more preferredembodiment, the alcohol is methanol. In another more preferredembodiment, the alcohol is 1,3-propanediol. In another more preferredembodiment, the alcohol is sorbitol. In another more preferredembodiment, the alcohol is xylitol. See, for example, Gong, C. S., Cao,N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewableresources, in Advances in Biochemical Engineering/Biotechnology,Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Silveira, M. M., and Jonas, R., 2002, The biotechnologicalproduction of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam,P., and Singh, D., 1995, Processes for fermentative production ofxylitol—a sugar substitute, Process Biochemistry, 30 (2): 117-124;Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production ofacetone, butanol and ethanol by Clostridium beijerinckii BA101 and insitu recovery by gas stripping, World Journal of Microbiology andBiotechnology 19 (6): 595-603.

In another preferred embodiment, the organic substance is an organicacid. In another more preferred embodiment, the organic acid is aceticacid. In another more preferred embodiment, the organic acid is acetonicacid. In another more preferred embodiment, the organic acid is adipicacid. In another more preferred embodiment, the organic acid is ascorbicacid. In another more preferred embodiment, the organic acid is citricacid. In another more preferred embodiment, the organic acid is2,5-diketo-D-gluconic acid. In another more preferred embodiment, theorganic acid is formic acid. In another more preferred embodiment, theorganic acid is fumaric acid. In another more preferred embodiment, theorganic acid is glucaric acid. In another more preferred embodiment, theorganic acid is gluconic acid. In another more preferred embodiment, theorganic acid is glucuronic acid. In another more preferred embodiment,the organic acid is glutaric acid. In another preferred embodiment, theorganic acid is 3-hydroxypropionic acid. In another more preferredembodiment, the organic acid is itaconic acid. In another more preferredembodiment, the organic acid is lactic acid. In another more preferredembodiment, the organic acid is malic acid. In another more preferredembodiment, the organic acid is malonic acid. In another more preferredembodiment, the organic acid is oxalic acid. In another more preferredembodiment, the organic acid is propionic acid. In another morepreferred embodiment, the organic acid is succinic acid. In another morepreferred embodiment, the organic acid is xylonic acid. See, forexample, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractivefermentation for lactic acid production from cellulosic biomass, Appl.Biochem. Biotechnol., 63-65: 435-448.

In another preferred embodiment, the organic substance is a ketone. Itwill be understood that the term “ketone” encompasses an organicsubstance that contains one or more ketone moieties. In another morepreferred embodiment, the ketone is acetone. See, for example, Qureshiand Blaschek, 2003, supra.

In another preferred embodiment, the organic substance is an amino acid.In another more preferred embodiment, the organic acid is aspartic acid.In another more preferred embodiment, the amino acid is glutamic acid.In another more preferred embodiment, the amino acid is glycine. Inanother more preferred embodiment, the amino acid is lysine. In anothermore preferred embodiment, the amino acid is serine. In another morepreferred embodiment, the amino acid is threonine. See, for example,Richard, A., and Margaritis, A., 2004, Empirical modeling of batchfermentation kinetics for poly(glutamic acid) production and othermicrobial biopolymers, Biotechnology and Bioengineering, 87 (4):501-515.

In another preferred embodiment, the organic substance is a gas. Inanother more preferred embodiment, the gas is methane. In another morepreferred embodiment, the gas is H₂. In another more preferredembodiment, the gas is CO₂. In another more preferred embodiment, thegas is CO. See, for example, Kataoka, N., A. Miya, and K. Kiriyama,1997, Studies on hydro-gen production by continuous culture system ofhydrogen-producing anaerobic bacteria, Water Science and Technology36(6-7): 41-47; and Gunaseelan V. N. in Biomass and Bioenergy, Vol. 13(1-2), pp. 83-114, 1997, Anaerobic digestion of biomass for methaneproduction: A review.

Production of an organic substance from lignocellulosic materialtypically requires four major steps. These four steps are pretreatment,enzymatic hydrolysis, fermentation, and recovery. Exemplified below is aprocess for producing ethanol, but it will be understood that similarprocesses can be used to produce other organic substances, for example,the substances described above.

Pretreatment. In the pretreatment or pre-hydrolysis step, thelignocellulosic material is heated to break down the lignin andcarbohydrate structure, solubilize most of the hemicellulose, and makethe cellulose fraction accessible to cellulolytic enzymes. The heatingis peformed either directly with steam or in slurry where a catalyst mayalso be added to the material to speed up the reactions. Catalystsinclude strong acids, such as sulfuric acid and SO₂, or alkali, such assodium hydroxide. The purpose of the pre-treatment stage is tofacilitate the penetration of the enzymes and microorganisms.Lignocellulosic biomass may also be subject to a hydrothermal steamexplosion pre-treatment (See U.S. Patent Application No. 20020164730).

Saccharification. In the enzymatic hydrolysis step, also known assaccharification, enzymes as described herein are added to thepretreated material to convert the cellulose fraction to glucose and/orother sugars. The saccharification is generally performed instirred-tank reactors or fermentors under controlled pH, temperature,and mixing conditions. A saccharification step may last up to 120 hours.Saccharification may be carried out at temperatures from about 30° C. toabout 65° C., in particular around 50° C., and at a pH in the rangebetween about 4 and about 5, especially around pH 4.5. To produceglucose that can be metabolized by yeast, the hydrolysis is typicallyperformed in the presence of a beta-glucosidase.

Fermentation. In the fermentation step, sugars, released from thelignocellulosic material as a result of the pretreatment and enzymatichydrolysis steps, are fermented to ethanol by a fermenting organism,such as yeast. The fermentation can also be carried out simultaneouslywith the enzymatic hydrolysis in the same vessel, again under controlledpH, temperature, and mixing conditions. When saccharification andfermentation are performed simultaneously in the same vessel, theprocess is generally termed simultaneous saccharification andfermentation or SSF.

Any suitable lignocellulosic substrate or raw material may be used in afermentation process of the present invention. The substrate isgenerally selected based on the desired fermentation product, i.e., theorganic substance to be obtained from the fermentation, and the processemployed, as is well known in the art. Examples of substrates suitablefor use in the methods of present invention, includelignocellulose-containing materials, such as wood or plant residues orlow molecular sugars DP₁₋₃ obtained from processed lignocellulosicmaterial that can be metabolized by the fermenting microorganism, andwhich may be supplied by direct addition to the fermentation medium.

The term “fermentation medium” will be understood to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism suitable for usein a desired fermentation process. Suitable fermenting microorganismsaccording to the invention are able to ferment, i.e., convert, sugars,such as glucose, xylose, arabinose, mannose, galactose, oroligosaccharides directly or indirectly into the desired fermentationproduct. Examples of fermenting microorganisms include fungal organisms,such as yeast. Preferred yeast includes strains of the Sacchromycesspp., and in particular, Sacchromyces cerevisiae. Commercially availableyeast include, e.g., Red Star®/Lesaffre Ethanol Red (available from RedStar/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a divisionof Burns Philp Food Inc., USA), SUPERSTART (available from Alltech),GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL(available from DSM Specialties).

In a preferred embodiment, the yeast is a Saccharomyces spp. In a morepreferred embodiment, the yeast is Saccharomyces cerevisiae. In anothermore preferred embodiment, the yeast is Saccharomyces distaticus. Inanother more preferred embodiment, the yeast is Saccharomyces uvarum. Inanother preferred embodiment, the yeast is a Kluyveromyces. In anothermore preferred embodiment, the yeast is Kluyveromyces marxianus. Inanother more preferred embodiment, the yeast is Kluyveromyces fragilis.In another preferred embodiment, the yeast is a Candida. In another morepreferred embodiment, the yeast is Candida pseudotropicalis. In anothermore preferred embodiment, the yeast is Candida brassicae. In anotherpreferred embodiment, the yeast is a Clavispora. In another morepreferred embodiment, the yeast is Clavispora lusitaniae. In anothermore preferred embodiment, the yeast is Clavispora opuntiae. In anotherpreferred embodiment, the yeast is a Pachysolen. In another morepreferred embodiment, the yeast is Pachysolen tannophilus. In anotherpreferred embodiment, the yeast is a Bretannomyces. In another morepreferred embodiment, the yeast is Bretannomyces clausenii (Philippidis,G. P., 1996, Cellulose bioconversion technology, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212).

Bacteria that can efficiently ferment glucose to ethanol include, forexample, Zymomonas mobilis and Clostridium thermocellum (Philippidis,1996, supra).

It is well known in the art that the various of the organisms describedabove can also be used to produce other organic substances, as describedherein.

The cloning of heterologous genes in Saccharomyces cerevisiae (Chen, Z.,Ho, N. W. Y., 1993, Cloning and improving the expression of Pichiastipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho, N. W. Y., Chen, Z, Brainard, A.P., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.,64: 1852-1859), or in bacteria such as Escherichia coli (Beall, D. S.,Ohta, K., Ingram, L. O., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303), Klebsiella oxytoca (Ingram, L. O., Gomes, P. F.,Lai, X., Moniruzzaman, M., Wood, B. E., Yomano, L. P., York, S. W.,1998, Metabolic engineering of bacteria for ethanol production,Biotechnol. Bioeng. 58: 204-214), and Zymomonas mobilis (Zhang, M.,Eddy, C., Deanda, K., Finkelstein, M., and Picataggio, S., 1995,Metabolic engineering of a pentose metabolism pathway in ethanologenicZymomonas mobilis, Science 267: 240-243; Deanda, K., Zhang, M., Eddy,C., and Picataggio, S., 1996, Development of an arabinose-fermentingZymomonas mobilis strain by metabolic pathway engineering, Appl.Environ. Microbiol. 62: 4465-4470) has led to the construction oforganisms capable of converting hexoses and pentoses to ethanol(cofermentation).

Yeast or another microorganism typically is added to the degradedlignocellulose or hydrolysate and the fermentation is ongoing for about24 to about 96 hours, such as about 35 to about 60 hours. Thetemperature is typically between about 26° C. to about 40° C., inparticular at about 32° C., and at about pH 3 to about pH 6, inparticular around pH 4-5.

In preferred embodiments, yeast or another microorganism is applied tothe degraded lignocellulose or hydrolysate and the fermentation isongoing for about 24 to about 96 hours, such as typically 35-60 hours.In preferred embodiments, the temperature is generally between about 26to about 40° C., in particular about 32° C., and the pH is generallyfrom about pH 3 to about pH 6, preferably around pH 4-5. Yeast oranother microorganism is preferably applied in amounts of approximately10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 5×10⁷ viable count per ml of fermentation broth. During anethanol producing phase the yeast cell count should preferably be in therange from approximately 10⁷ to 10¹⁰, especially around approximately2×10⁸. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

The most widely used process in the art is the simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzymeare added together.

For ethanol production, following the fermentation the mash is distilledto extract the ethanol. The ethanol obtained according to the process ofthe invention may be used as, e.g., fuel ethanol; drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator may be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, e.g., Alfenore et al.,Improving ethanol production and viability of Saccharomyces cerevisiaeby a vitamin feeding strategy during fed-batch process,” Springer-Verlag(2002), which is hereby incorporated by reference. Examples of mineralsinclude minerals and mineral salts that can supply nutrients comprisingP, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Recovery. The alcohol is separated from the fermented lignocellulosicmaterial and purified by conventional methods of distillation. Ethanolwith a purity of up to about 96 vol. % ethanol can be obtained, whichcan be used as, for example, fuel ethanol, drinking ethanol, i.e.,potable neutral spirits, or industrial ethanol.

For other organic substances, any method known in the art can be usedincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, distillation, or extraction.

Additional Enzymes

In the methods of the present invention, the cellulolytic enzyme(s) maybe supplemented by one or more additional enzyme activities to improvethe degradation of the lignocellulosic material. Preferred additionalenzymes are hemicellulases, esterases (e.g., lipases, phospholipases,and/or cutinases), proteases, laccases, peroxidases, or mixturesthereof.

In the methods of the present invention, the additional enzyme(s) may beadded prior to or during fermentation, including during or after thepropagation of the fermenting microorganism(s).

The enzymes referenced herein may be derived or obtained from anysuitable origin, including, bacterial, fungal, yeast or mammalianorigin. The term “obtained” means herein that the enzyme may have beenisolated from an organism which naturally produces the enzyme as anative enzyme. The term “obtained” also means herein that the enzyme mayhave been produced recombinantly in a host organism, wherein therecombinantly produced enzyme is either native or foreign to the hostorganism or has a modified amino acid sequence, e.g., having one or moreamino acids which are deleted, inserted and/or substituted, i.e., arecombinantly produced enzyme which is a mutant and/or a fragment of anative amino acid sequence or an enzyme produced by nucleic acidshuffling processes known in the art. Encompassed within the meaning ofa native enzyme are natural variants and within the meaning of a foreignenzyme are variants obtained recombinantly, such as by site-directedmutagenesis or shuffling.

The enzymes may also be purified. The term “purified” as used hereincovers enzymes free from other components from the organism from whichit is derived. The term “purified” also covers enzymes free fromcomponents from the native organism from which it is obtained. Theenzymes may be purified, with only minor amounts of other proteins beingpresent. The expression “other proteins” relate in particular to otherenzymes. The term “purified” as used herein also refers to removal ofother components, particularly other proteins and most particularlyother enzymes present in the cell of origin of the enzyme of theinvention. The enzyme may be “substantially pure,” that is, free fromother components from the organism in which it is produced, that is, forexample, a host organism for recombinantly produced enzymes. Inpreferred embodiment, the enzymes are at least 75% (w/w), preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 95%, more preferably at least 96%, morepreferably at least 97%, even more preferably at least 98%, or mostpreferably at least 99% pure. In another preferred embodiment, theenzyme is 100% pure.

The enzymes used in the present invention may be in any form suitablefor use in the processes described herein, such as, for example, in theform of a dry powder or granulate, a non-dusting granulate, a liquid, astabilized liquid, or a protected enzyme. Granulates may be produced,e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452, and mayoptionally be coated by process known in the art. Liquid enzymepreparations may, for instance, be stabilized by adding stabilizers suchas a sugar, a sugar alcohol or another polyol, and/or lactic acid oranother organic acid according to established process. Protected enzymesmay be prepared according to the process disclosed in EP 238,216.

Hemicellulases

Enzymatic hydrolysis of hemicelluloses can be performed by a widevariety of fungi and bacteria. Similar to cellulose degradation,hemicellulose hydrolysis requires coordinated action of many enzymes.Hemicellulases can be placed into three general categories: theendo-acting enzymes that attack internal bonds within the polysaccharidechain, the exo-acting enzymes that act processively from either thereducing or nonreducing end of polysaccharide chain, and the accessoryenzymes, acetylesterases and esterases that hydrolyze lignin glycosidebonds, such as coumaric acid esterase and ferulic acid esterase (Wong,K. K. Y., Tan, L. U. L., and Saddler, J. N., 1988, Multiplicity ofβ-1,4-xylanase in microorganisms: Functions and applications, Microbiol.Rev. 52: 305-317; Tenkanen, M., and Poutanen, K., 1992, Significance ofesterases in the degradation of xylans, in Xylans and Xylanases, Visser,J., Beldman, G., Kuster-van Someren, M. A., and Voragen, A. G. J., eds.,Elsevier, New York, N.Y., 203-212; Coughlan, M. P., and Hazlewood, G.P., 1993, Hemicellulose and hemicellulases, Portland, London, UK;Brigham, J. S., Adney, W. S., and Himmel, M. E., 1996, Hemicellulases:Diversity and applications, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,119-141).

Hemicellulases include xylanases, arabinofuranosidases, acetyl xylanesterase, glucuronidases, endo-galactanase, mannanases, endo or exoarabinases, exo-galactanses, and mixtures thereof. Examples ofendo-acting hemicellulases and ancillary enzymes includeendoarabinanase, endoarabinogalactanase, endoglucanase, endomannanase,endoxylanase, and feraxan endoxylanase. Examples of exo-actinghemicellulases and ancillary enzymes include α-L-arabinosidase,β-L-arabinosidase, α-1,2-L-fucosidase, α-D-galactosidase,β-D-galactosidase, β-D-glucosidase, β-D-glucuronidase, β-D-mannosidase,β-D-xylosidase, exoglucosidase, exocellobiohydrolase,exomannobiohydrolase, exomannanase, exoxylanase, xylan α-glucuronidase,and coniferin β-glucosidase. Examples of esterases include acetylesterases (acetylgalactan esterase, acetylmannan esterase, andacetylxylan esterase) and aryl esterases (coumaric acid esterase andferulic acid esterase).

Preferably, the hemicellulase is an exo-acting hemicellulase, and morepreferably, an exo-acting hemicellulase which has the ability tohydrolyze hemicellulose under acidic conditions of below pH 7. Anexample of a hemicellulase suitable for use in the present inventionincludes VISCOZYME™ (available from Novozymes A/S, Denmark). Thehemicellulase is added in an effective amount from about 0.001% to about5.0% wt. of solids, more preferably from about 0.025% to about 4.0% wt.of solids, and most preferably from about 0.005% to about 2.0% wt. ofsolids.

A xylanase (E.C. 3.2.1.8) may be obtained from any suitable source,including fungal and bacterial organisms, such as Aspergillus,Disporotrichum, Penicillium, Neurospora, Fusarium, Trichoderma,Humicola, Thermomyces, and Bacillus. Preferred commercially availablepreparations comprising xylanase include SHEARZYME®, BIOFEED WHEAT®,BIO-FEED Plus® L, CELLUCLAST®, ULTRAFLO®, VISCOZYME®, PENTOPAN MONO® BG,and PULPZYME® HC (Novozymes A/S); and LAMINEX® and SPEZYME® CP (GenencorInt.).

Esterases

Esterases that can be used for bioconversion of lignocellulose includeacetyl esterases such as acetylgalactan esterase, acetylmannan esterase,and acetylxylan esterase, and esterases that hydrolyze lignin glycosidebonds, such as coumaric acid esterase and ferulic acid esterase.

As used herein, an “esterase” also known as a carboxylic esterhydrolyase, refers to enzymes acting on ester bonds, and includesenzymes classified in EC 3.1.1 Carboxylic Ester Hydrolases according toEnzyme Nomenclature (Enzyme Nomenclature 1992, Academic Press, SanDiego, Calif., with Supplement 1 (1993), Supplement 2 (1994), Supplement3 (1995), Supplement 4 (1997) and Supplement 5, in Eur. J. Biochem. 223:1-5, 1994; Eur. J. Biochem. 232: 1-6,1995; Eur. J. Biochem. 237: 1-5,1996; Eur. J. Biochem. 250: 1-6, 1997, and Eur. J. Biochem. 264:610-650, 1999; respectively). Non-limiting examples of esterases includearylesterase, triacylglycerol lipase, acetylesterase,acetylcholinesterase, cholinesterase, tropinesterase, pectinesterase,sterol esterase, chlorophyllase, L-arabinonolactonase, gluconolactonase,uronolactonase, tannase, retinyl-palmitate esterase,hydroxybutyrate-dimer hydrolase, acylglycerol lipase, 3-oxoadipateenol-lactonase, 1,4-lactonase, galactolipase, 4-pyridoxolactonase,acylcarnitine hydrolase, aminoacyl-tRNA hydrolase, D-arabinonolactonase,6-phosphogluconolactonase, phospholipase A1, 6-acetylglucosedeacetylase, lipoprotein lipase, dihydrocoumarin lipase,limonin-D-ring-lactonase, steroid-lactonase, triacetate-lactonase,actinomycin lactonase, orsellinate-depside hydrolase, cephalosporin-Cdeacetylase, chlorogenate hydrolase, alpha-amino-acid esterase,4-methyloxaloacetate esterase, carboxymethylenebutenolidase,deoxylimonate A-ring-lactonase, 2-acetyl-1-alkylglycerophosphocholineesterase, fusarinine-C ornithinesterase, sinapine esterase, wax-esterhydrolase, phorbol-diester hydrolase, phosphatidylinositol deacylase,sialate O-acetylesterase, acetoxybutynylbithiophene deacetylase,acetylsalicylate deacetylase, methylumbelliferyl-acetate deacetylase,2-pyrone-4,6-dicarboxylate lactonase, N-acetylgalactosaminoglycandeacetylase, juvenile-hormone esterase, bis(2-ethylhexyl)phthalateesterase, protein-glutamate methylesterase, 11-cis-retinyl-palmitatehydrolase, all-trans-retinyl-palmitate hydrolase,L-rhamnono-1,4-lactonase, 5-(3,4-diacetoxybut-1-ynyl)-2,2′-bithiophenedeacetylase, fatty-acyl-ethyl-ester synthase, xylono-1,4-lactonase,N-acetylglucosaminylphosphatidylinositol deacetylase, cetraxatebenzylesterase, acetylalkylglycerol acetylhydrolase, and acetylxylanesterase.

Preferred esterases for use in the present invention are lipolyticenzymes, such as, lipases (classified as EC 3.1.1.3, EC 3.1.1.23, and/orEC 3.1.1.26) and phospholipases (classified as EC 3.1.1.4 and/or EC3.1.1.32, including lysophospholipases classified as EC 3.1.1.5). Otherpreferred esterases are cutinases (classified as EC 3.1.1.74).

The esterase may be added in an amount effective to obtain the desiredbenefit to improve the performance of the fermenting microorganism, forexample, to change the lipid composition/concentration inside and/oroutside of the fermenting microorganism or in the cell membrane of thefermenting microorganism, to result in an improvement in the movement ofsolutes into and/or out of the fermenting microorganisms duringfermentation and/or to provide more metabolizable energy sources (suchas, for example, by converting components, such as, oil from the cornsubstrate, to components useful the fermenting microorganism, e.g.,unsaturated fatty acids and glycerol), to increase ethanol yield.Examples of effective amounts of esterase are from about 0.01 to about400 LU/g DS (Dry Solids). Preferably, the esterase is used in an amountof about 0.1 to about 100 LU/g DS, more preferably about 0.5 to about 50LU/g DS, and even more preferably about 1 to about 20 LU/g DS. Furtheroptimization of the amount of esterase can hereafter be obtained usingstandard procedures known in the art.

One Lipase Unit (LU) is the amount of enzyme which liberates 1.0 μmol oftitratable fatty acid per minute with tributyrin as substrate and gumarabic as an emulsifier at 30° C., pH 7.0 (phosphate buffer).

In a preferred embodiment, the esterase is a lipolytic enzyme, morepreferably, a lipase. As used herein, a “lipolytic enzyme” refers tolipases and phospholipases (including lyso-phospholipases). Thelipolytic enzyme is preferably of microbial origin, in particular ofbacterial, fungal or yeast origin. The lipolytic enzyme used may bederived from any source, including, for example, a strain of Absidia, inparticular Absidia blakesleena and Absidia corymbifera, a strain ofAchromobacter, in particular Achromobacter iophagus, a strain ofAeromonas, a strain of Alternaria, in particular Alternaria brassiciola,a strain of Aspergillus, in particular Aspergillus niger, Aspergillusoryzae, Asoergillus fumigatus, and Aspergillus flavus, a strain ofAchromobacter, in particular Achromobacter iophagus, a strain ofAureobasidium, in particular Aureobasidium pullulans, a strain ofBacillus, in particular Bacillus pumilus, Bacillus stearothermophilus,and Bacillus subtilis, a strain of Beauveria, a strain of Brochothrix,in particular Brochothrix thermosohata, a strain of Candida, inparticular Candida cylindracea (Candida rugosa), Candida paralipolytica,and Candida antarctica, a strain of Chromobacter, in particularChromobacter viscosum, a strain of Coprinus, in particular Coprinuscinerius, a strain of Fusarium, in particular Fusarium graminearum,Fusarium oxysporum, Fusarium solani, Fusarium solani pisi, Fusariumroseum culmorum, and Fusarium venenatum, a strain of Geotricum, inparticular Geotricum penicillatum, a strain of Hansenula, in particularHansenula anomala, a strain of Humicola, in particular Humicolabrevispora, Humicola brevis var. thermoidea, and Humicola insolens, astrain of Hyphozyma, a strain of Lactobacillus, in particularLactobacillus curvatus, a strain of Metarhizium, a strain of Mucor, astrain of Paecilomyces, a strain of Penicillium, in particularPenicillium cyclopium, Penicillium crustosum and Penicillium expansum, astrain of Pseudomonas in particular Pseudomonas aeruginosa, Pseudomonasalcaligenes, Pseudomonas cepacia (syn. Burkholderia cepacia),Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas maltophilia,Pseudomonas mendocina, Pseudomonas mephitica lipolytica, Pseudomonasalcaligenes, Pseudomonas plantari, Pseudomonas pseudoalcaligenes,Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonaswisconsinensis, a strain of Rhizooctonia, in particular Rhizooctoniasolani, a strain of Rhizomucor, in particular Rhizomucor miehei, astrain of Rhizopus, in particular Rhizopus japonicus, Rhizopusmicrosporus, and Rhizopus nodosus, a strain of Rhodosporidium, inparticular Rhodosporidium toruloides, a strain of Rhodotorula, inparticular Rhodotorula glutinis, a strain of Sporobolomyces, inparticular Sporobolomyces shibatanus, a strain of Thermomyces, inparticular Thermomyces lanuginosus (formerly Humicola lanuginosa), astrain of Thiarosporella, in particular Thiarosporella phaseolina, astrain of Trichoderma, in particular, Trichoderma harzianum andTrichoderma reesei, and/or a strain of Verticillium.

In a preferred embodiment, the lipolytic enzyme is derived from a strainof Aspergillus, Achromobacter, Bacillus, Candida, Chromobacter,Fusarium, Humicola, Hyphozyma, Pseudomonas, Rhizomucor, Rhizopus, orThermomyces.

In more preferred embodiments, the lipolytic enzyme is a lipase. Lipasesmay be applied herein for their ability to modify the structure andcomposition of triglyceride oils and fats in the fermentation media(including fermentation yeast), for example, resulting from a cornsubstrate. Lipases catalyze different types of triglyceride conversions,such as hydrolysis, esterification, and transesterification. Suitablelipases include acidic, neutral, and basic lipases, as are well-known inthe art, although acidic lipases (such as, e.g., the lipase G AMANO 50,available from Amano) appear to be more effective at lowerconcentrations of lipase as compared to either neutral or basic lipases.Preferred lipases for use in the present invention include Candidaantarcitca lipase and Candida cylindracea lipase. More preferred lipasesare purified lipases such as Candida antarcitca lipase (lipase A),Candida antarcitca lipase (lipase B), Candida cylindracea lipase, andPenicillium camembertii lipase.

The lipase may be the one disclosed in EP 258,068-A or may be a lipasevariant such as a variant disclosed in WO 00/60063 or WO 00/32758,hereby incorporated by reference. Preferred commercial lipases includeLECITASE™, LIPOLASE™, and LIPEX™ (available from Novozymes A/S, Denmark)and G AMANO™ 50 (available from Amano).

Lipases are preferably added in amounts from about 1 to about 400 LU/gDS, preferably about 1 to about 10 LU/g DS, and more preferably about 1to about 5 LU/g DS.

In another preferred embodiment of the present invention, the esteraseis a cutinase. Cutinases are enzymes which are able to degrade cutin.The cutinase may be derived from any source. In a preferred embodiment,the cutinase is derived from a strain of Aspergillus, in particularAspergillus oryzae, a strain of Alternaria, in particular Alternariabrassiciola, a strain of Fusarium, in particular Fusarium solani,Fusarium solani pisi, Fusarium roseum culmorum, or Fusarium roseumsambucium, a strain of Helminthosporum, in particular Helminthosporumsativum, a strain of Humicola, in particular Humicola insolens, a strainof Pseudomonas, in particular Pseudomonas mendocina or Pseudomonasputida, a strain of Rhizoctonia, in particular Rhizoctonia solani, astrain of Streptomyces, in particular Streptomyces scabies, or a strainof Ulocladium, in particular Ulocladium consortiale. In a most preferredembodiment the cutinase is derived from a strain of Humicola insolens,in particular the strain Humicola insolens DSM 1800. Humicola insolenscutinase is described in WO 96/13580, which is hereby incorporated byreference. The cutinase may be a variant such as one of the variantsdisclosed in WO 00/34450 and WO 01/92502, which are hereby incorporatedby reference. Preferred cutinase variants include variants listed inExample 2 of WO 01/92502 which are hereby specifically incorporated byreference. An effective amount of cutinase is from about 0.01 to about400 LU/g DS, preferably from about 0.1 to about 100 LU/g DS, and morepreferably from about 1 to about 50 LU/g DS. Further optimization of theamount of cutinase can hereafter be obtained using standard proceduresknown in the art.

In another preferred embodiment, the esterase is a phospholipase. Asused herein, the term “phospholipase” is an enzyme which has activitytowards phospholipids, e.g., hydrolytic activity. Phospholipids, such aslecithin or phosphatidylcholine, consist of glycerol esterified with twofatty acids in an outer (sn-1) and the middle (sn-2) positions andesterified with phosphoric acid in the third position. The phosphoricacid may be esterified to an amino-alcohol. Several types ofphospholipase activity can be distinguished, including phospholipases A₁and A₂ which hydrolyze one fatty acyl group (in the sn-1 and sn-2position, respectively) to form lysophospholipid; and lysophospholipase(or phospholipase B) which hydrolyzes the remaining fatty acyl group inlysophospholipid. Phospholipase C and phospholipase D(phosphodiesterases) release diacyl glycerol or phosphatidic acidrespectively.

The term “phospholipase” includes enzymes with phospholipase activity,e.g., phospholipase A (A₁ or A₂), phospholipase B activity,phospholipase C activity, or phospholipase D activity. The term“phospholipase A” as used herein is intended to cover an enzyme withphospholipase A₁ and/or phospholipase A₂ activity. The phospholipaseactivity may be provided by enzymes having other activities as well,such as, e.g., a lipase with phospholipase activity. The phospholipaseactivity may, for example, be from a lipase with phospholipase sideactivity. In other embodiments, the phospholipase enzyme activity isprovided by an enzyme having essentially only phospholipase activity andwherein the phospholipase enzyme activity is not a side activity.

The phospholipase may be of any origin, for example, of animal origin(e.g., mammalian, for example, bovine or porcine pancreas), or snakevenom or bee venom. Alternatively, the phospholipase may be of microbialorigin, for example, from filamentous fungi, yeast or bacteria, such asAspergillus, e.g., A. awamori, A. foetidus, A. japonicus, A. niger, orA. oryzae, Dictyostelium, e.g., D. discoideum; Fusarium, e.g., F.culmorum, F. graminearum, F. heterosporum, F. solani, F. oxysporum, orF. venenatum; Mucor, e.g., M. javanicus, M. mucedo, or M. subtilissimus;Neurospora, e.g., N. crassa; Rhizomucor, e.g., R. pusillus; Rhizopus,e.g., R. arrhizus, R. japonicus, R. stolonifer, Sclerotinia, e.g., S.libertiana; Trichophyton, e.g., T. rubrum; Whetzelinia, e.g., W.sclerotiorum; Bacillus, e.g., B. megaterium, or B. subtilis;Citrobacter, e.g., C. freundii; Enterobacter, e.g., E. aerogenes or E.cloacae; Edwardsiella, E. tarda; Erwinia, e.g., E. herbicola;Escherichia, e.g., E. coli; Klebsiella, e.g., K. pneumoniae; Proteus,e.g., P. vulgaris; Providencia, e.g., P. stuartii; Salmonella, e.g., S.typhimurium; Serratia, e.g., S. liquefasciens, S. marcescens; Shigella,e.g., S. flexneri; Streptomyces, e.g., S. violeceoruber; or Yersinia,e.g., Y. enterocolitica.

Preferred commercial phospholipases include LECITASE™ and LECITASE™ULTRA (available from Novozymes A/S, Denmark).

An effective amount of phospholipase is from about 0.01 to about 400LU/g DS, preferably from about 0.1 to about 100 LU/g DS, and morepreferably from about 1 to about 50 LU/g DS. Further optimization of theamount of phospholipase can hereafter be obtained using standardprocedures known in the art.

Proteases

In another preferred embodiment of the invention, at least onesurfactant and at least one carbohydrate generating enzyme is used incombination with at least one protease. The protease may be used, e.g.,to digest protein to produce free amino nitrogen (FAN). Such free aminoacids function as nutrients for the yeast, thereby enhancing the growthof the yeast and, consequently, the production of ethanol.

The fermenting microorganism for use in a fermentation process may beproduced by propagating the fermenting microorganism in the presence ofat least one protease. Although not limited to any one theory ofoperation, it is believed that the propagation of the fermentingmicroorganism with an effective amount of at least one protease reducesthe lag time of the fermenting microorganism when the fermentingmicroorganism is subsequently used in a fermentation process as comparedto a fermenting microorganism that was propogated under the sameconditions without the addition of the protease. The action of theprotease in the propagation process is believed to directly orindirectly result in the suppression or expression of genes which aredetrimental or beneficial, respectively, to the fermenting microorganismduring fermentation, thereby decreasing lag time and resulting in afaster fermentation cycle.

Proteases are well known in the art and refer to enzymes that catalyzethe cleavage of peptide bonds. Suitable proteases include fungal andbacterial proteases. Preferred proteases are acidic proteases, i.e.,proteases characterized by the ability to hydrolyze proteins underacidic conditions below pH 7. Suitable acid fungal proteases includefungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida,Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotium, andTorulopsis. Especially contemplated are proteases derived fromAspergillus niger (see, e.g., Koaze et al., 1964, Agr. Biol. Chem. Japan28: 216), Aspergillus saitoi (see, e.g., Yoshida, 1954, J. Agr. Chem.Soc. Japan 28: 66), Aspergillus awamori (Hayashida et al., 1977, Agric.Biol. Chem. 42: 927-933, Aspergillus aculeatus (WO 95/02044), orAspergillus oryzae; and acidic proteases from Mucor pusillus or Mucormiehei.

Bacterial proteases, which are not acidic proteases, include thecommercially available products ALCALASE™ and NEUTRASE™ (available fromNovozymes A/S). Other proteases include GC106 from Genencor Int, Inc.,USA and NOVOZYM™ 50006 from Novozymes A/S.

Preferably, the protease is an aspartic acid protease, as described, forexample, in Handbook of Proteolytic Enzymes, Edited by A. J. Barrett, N.D. Rawlings and J. F. Woessner, Academic Press, San Diego, 1998, Chapter270). Suitable examples of aspartic acid protease include, e.g., thosedisclosed by Berka et al., 1990, Gene 96: 313; Berka et al., 1993, Gene125: 195-198; and Gomi et al., 1993, Biosci. Biotech. Biochem. 57:1095-1100.

Peroxidases

Other compounds possessing peroxidase activity may be any peroxidase (EC1.11.1.7), or any fragment having peroxidase activity derived therefrom,exhibiting peroxidase activity.

Preferably, the peroxidase is produced by plants (e.g., horseradish orsoybean peroxidase) or microorganisms such as fungi or bacteria.

Some preferred fungi include strains belonging to the subdivisionDeuteromycotina, class Hyphomycetes, e.g., Fusarium, Humicola,Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces,Ulocladium, Embellisia, Cladosporium, or Dreschlera, in particularFusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii,Myrothecium verrucaria (IFO 6113), Verticillum alboatrum, Verticillumdahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago,Ulocladium chartarum, Embellisia alli, or Dreschlera halodes.

Other preferred fungi include strains belonging to the subdivisionBasidiomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete,Coriolus, or Trametes, in particular Coprinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.NA-12), or Trametes (previously called Polyporus), e.g., T. versicolor(e.g. PR4 28-A).

Further preferred fungi include strains belonging to the subdivisionZygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, in particularMucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales,e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus(IFO 12382), or Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonaspalustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958),Pseudomonas fluorescens (NRRL B-11), and Bacillus strains, e.g.,Bacillus pumilus (ATCC 12905) and Bacillus stearothermophilus.

Further preferred bacteria include strains belonging to Myxococcus,e.g., M. virescens.

The peroxidase may also be one which is produced by a method comprisingcultivating a host cell transformed with a recombinant DNA vector whichcarries a DNA sequence encoding the peroxidase as well as DNA sequencesfor expression of the DNA sequence encoding the peroxidase, in a culturemedium under conditions permitting the expression of the peroxidase andrecovering the peroxidase from the culture.

In a preferred embodiment, a recombinantly produced peroxidase is aperoxidase derived from a Coprinus sp., in particular C. macrorhizus orC. cinereus according to WO 92/16634.

In the present invention, compounds possessing peroxidase activitycomprise peroxidase enzymes and peroxidase active fragments derived fromcytochromes, haemoglobin, or peroxidase enzymes.

One peroxidase unit (POXU) is the amount of enzyme which under thefollowing conditions catalyzes the conversion of 1 mmole hydrogenperoxide per minute at 30° C. in 0.1 M phosphate buffer pH 7.0, 0.88 mMhydrogen peroxide, and 1.67 mM2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). The reactionis followed for 60 seconds (15 seconds after mixing) by the change inabsorbance at 418 nm, which should be in the range of 0.15 to 0.30. Forcalculation of activity, an absorption coefficient of oxidized ABTS of36 mM⁻¹ cm⁻¹ and a stoichiometry of one pmole H₂O₂ converted per twopmole ABTS oxidized are used.

Laccases

In the present invention, laccases and laccase related enzymes compriseany laccase enzyme classified as EC 1.10.3.2, any catechol oxidaseenzyme classified as EC 1.10.3.1, any bilirubin oxidase enzymeclassified as EC 1.3.3.5, or any monophenol monooxygenase enzymeclassified as EC 1.14.18.1.

The above-mentioned enzymes may be microbial, i.e., obtained frombacteria or fungi (including filamentous fungi and yeasts), or they maybe derived from plants.

Suitable examples from fungi include a laccase obtained from a strain ofAspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia,Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizooctonia, e.g., R. solani, Coprinus, e.g., C. cinereus,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Pycnoporus, e.g., P. cinnabarinus, Phlebia, e.g., P. radita(WO 92/01046), or Coriolus, e.g., C. hirsutus (JP 2-238885).

Suitable examples from bacteria include a laccase obtained from a strainof Bacillus.

A laccase obtained from Coprinus, Myceliophthora, Polyporus, Pycnoporus,Scytalidium or Rhizoctonia is preferred; in particular a laccaseobtained from Coprinus cinereus, Myceliophthora thermophila, Polyporuspinsitus, Pycnoporus cinnabarinus, Scytalidium thermophilum, orRhizoctonia solani.

Commercially available laccases are NS51001 (a Polyporus pinsitiuslaccase, available from Novozymes A/S, Denmark) and NS51002 (aMyceliopthora thermophila laccase, available from Novozymes A/S,Denmark).

The laccase or the laccase related enzyme may also be one which isproduced by a method comprising cultivating a host cell transformed witha recombinant DNA vector which carries a DNA sequence encoding thelaccase as well as DNA sequences for expression of the DNA sequenceencoding the laccase, in a culture medium under conditions permittingthe expression of the laccase enzyme, and recovering the laccase fromthe culture.

Laccase activity (LACU) is determined from the oxidation ofsyringaldazin under aerobic conditions at pH 5.5. The violet colourproduced is photometered at 530 nm. The analytical conditions are 19 mMsyringaldazin, 23 mM acetate buffer, pH 5.5, 30° C., 1 minute reactiontime. One laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazin per minute under the aboveconditions.

Laccase activity (LAMU) is determined from the oxidation ofsyringaldazin under aerobic conditions at pH 7.5. The violet colourproduced is photometered at 530 nm. The analytical conditions are 19 mMsyringaldazin, 23 mM Tris/maleate pH 7.5, 30° C., 1 minute reactiontime. One laccase unit (LAMU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazin per minute under the aboveconditions.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Trichoderma reesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979,Adv. Chem. Ser. 181: 289-301), which was derived from Trichoderma reeseiQm6A (ATCC 13631; Mandels and Reese, 1957, J. Bacteriol. 73: 269-278),was used as a host for the expression of Aspergillus oryzaebeta-glucosidase.

Escherichia coli strain TOP10 cells (Invitrogen, Carlsbad, Calif.) andEpicurian coli SURE electroporation-competent cells (Stratagene, LaJolla, Calif.) were used for propagation of plasmids.

Aspergillus oryzae JaL250 strain (WO 99/61651) was used for expressionof the Aspergillus fumigatus beta-glucosidase. Aspergillus fumigatusPaHa34 was used as the source of the Family GH3A beta-glucosidase.

Media and Buffer Solutions

YP medium was composed per liter of 10 g of yeast extract and 20 g ofbactopeptone.

COVE selection plates were composed per liter of 342.3 g of sucrose, 20ml of COVE salt solution, 10 mM acetamide, 15 mM CsCl₂, and 25 g ofNoble agar.

COVE2 plates were composed per liter of 30 g of sucrose, 20 ml COVE ofsalt solution, 10 mM acetamide, and 25 g of Noble agar.

COVE salt solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals solution.

COVE trace metals solution was composed per liter of 0.04 g ofNaB₄07110H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

Cellulase-inducing media was composed per liter of 20 g of ArbocelB800-natural cellulose fibers (J. Rettenmaier USA LP, Schoolcraft,Mich.), 10 g of corn steep solids (Sigma Chemical Co., St. Louis, Mo.),1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g of CaCl₂, 0.42 g ofMgSO₄.7H₂O, 0.42 ml Trichoderma reesei trace metals solution, and 2drops of pluronic acid; pH to 6.0 with 10 N NaoH.

Trichoderma reesei trace metals solution was composed per liter of 216 gof FeCl₃.6H₂O, 58 g of ZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g ofCuSO₄.5H₂O, 2.4 g of H₃BO₃, and 336 g of citric acid.

PEG Buffer was composed per liter of 500 g of PEG 4000 (BDH, Poole,England), 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5 (filter sterilize).

STC was composed per liter of 1 M sorbitol, 10 mM CaCl₂, and 10 mMTris-HCl pH 7.5 (filter sterilize).

YPD medium was composed per liter of 10 g of yeast extract, 20 g ofbacto tryptone, and 40 ml of 50% glucose.

Yeast selection medium was composed per liter of 6.7 g of yeast nitrogenbase, 0.8 g of complete supplement mixture (CSM, Qbiogene, Inc.,Carlsbad, Calif.; missing uracil and containing 40 mg/ml of adenine), 5g of casamino acids (without amino acids), 100 ml of 0.5 M succinate pH5.0, 40 ml of 50% glucose, 1 ml of 100 mM CUSO₄, 50 mg of ampicillin,and 25 mg of chloramphenicol.

Yeast selection plate medium was composed per liter of yeast selectionmedium supplemented with 20 g of bacto agar and 150 mg of5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside (X-Glc, INALCO SPA,Milano, Italy) but lacking both ampicillin and chloramphenicol.

Potato dextrose medium was composed per liter of 39 grams of potatodextrose (Difco).

PDA plates were composed per liter of 39 grams of potato dextrose agar.

MDU2BP medium was composed per liter of 45 g of maltose, 1 g ofMgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeastextract, 2 g of urea, and 0.5 ml of AMG trace metals solution, pH to5.0.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, and 3 g of citric acid.

CIM medium was composed per liter of 20 g of cellulose, 10 g of cornsteep solids, 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g of CaCl₂,0.42 g of MgSO₄.7H₂O, and 0.42 ml of trace metals solution, pH to 6.0.

Trace metals solution was composed per liter of 41.2 mg of FeCl₃.6H₂O,11.6 mg of ZnSO₄.7H₂O, 5.4 mg of MnSO₄.H₂O, 2.0 mg of CuSO₄.5H₂O, 0.48mg of H₃BO₃, and 67.2 mg of citric acid.

Beta-Glucosidase Activity Assay

Beta-glucosidase activity was determined at ambient temperature on 25 μlof crude culture supernatants, diluted 1:10 in 50 mM succinate pH 5.0,using 200 μl of 0.5 mg/ml p-nitrophenyl-beta-D-glucopyranoside (pnpBDG)as substrate in 50 mM succinate pH 5.0. After 15 minutes incubation thereaction was stopped by adding 100 μl of 1 M Tris-HCl pH 8.0 and theabsorbance was read spectrophotometrically at 405 nm. Aspergillus nigerbeta-glucosidase (Novozyme 188, Novozymes A/S, Bagsveerd, Denmark) wasused as an enzyme standard.

Endoglucanase Activity Assay

The specific activity of endoglucanases towards carboxymethylcellulose(CMC, 5 mg/ml) was determined by measuring the initial rate ofhydrolysis in the range of linear increase of reducing sugar (RS)concentration over time in 50 mM sodium acetate pH 5.0 at 50° C.Hydrolysis was carried out without stirring in the presence of 0.5 mg/mlBSA. Specific activity was expressed in international units (IU) per mgprotein. One IU is defined as the μmol of glycosidic bonds hydrolyzed inone minute during the initial period of hydrolysis. Enzymes were dilutedso as to give a linear relationship between enzyme concentration andactivity measured.

Carboxymethylcellulose (type 7L2, Hercules Inc., Wilmington, Del.) withaverage degree of substitution (DS) of 0.7. A 6.25 mg/ml solution of CMCin 50 mM sodium acetate pH 5.0 was prepared by slowly adding CMC tovigorously agitated buffer, followed by heating to approximately 60° C.under continuous stirring until complete dissolution.

Cellobiohydrolase Activity Assay

Cellobiohydrolase activity was determined according to the proceduredescribed by Deshpande et al. (Deshpande, M. V., Eriksson, K.-E.,Pettersson, L. G., 1984, An assay for selective determination ofexo-1,4-β-glucanases in a mixture of cellulolytic enzymes, Anal.Biochem. 138: 481-487), which was modified to a 96-well microplateformat. Concentration of p-nitrophenol (PNP) produced from 2.5 mMp-nitrophenyl-β-D-cellobioside (PNPC, Cat. # 5754, Sigma, St. Louis,Mo.) was measured spectrophotometrically at 405 nm (A₄₀₅) after 30minutes of hydrolysis in 50 mM sodium acetate pH 5.0 at 40° C. Prior tohydrolysis, the enzymes were diluted in 50 mM sodium acetate pH 5.0 togive less than 8% conversion of PNPC at the conditions specified.

Enzymes

Celluclast 1.5 L was obtained from Novozymes A/S, Bagsvaerd, Denmark.Protein concentration in cellulase samples was determined by the BCAMicroplate Assay as described in the Instructions for PCS Protein AssayReagent Kit (Pierce Chemical Co., Rockford, Ill.). Prior to determiningthe concentration, some samples were desalted by passing through BioSpin 6 columns (BioRad Laboratories, Hercules, Calif.) according to themanufacturer's instructions.

Prior to hydrolysis experiments, Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO02/095014), Aspergillus fumigatis beta-glucosidase (recombinantlyproduced in Aspergillus oryzae), and Trichoderma reesei endoglucanase I(recombinantly produced in Aspergillus oryzae) were desalted andexchanged into 50 mM sodium acetate pH 5.0 buffer, using CentriconPlus-20 centrifugal filter with Biomax-5 membrane (5000 NMWL; Millipore,Bedford, Mass.).

Enzyme dilutions were prepared fresh before each experiment from stockenzyme solutions, which were stored at −20° C.

Example 1 Construction of pAILo1 Expression Vector

Expression vector pAILo1 was constructed by modifying pBANe6 (U.S. Pat.No. 6,461,837), which comprises a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase (NA2-tpi promoter), Aspergillus nigeramyloglucosidase terminator sequence (AMG terminator), and Aspergillusnidulans acetamidase gene (amdS). Modification of pBANe6 was performedby first eliminating three Nco I restriction sites at positions 2051,2722, and 3397 bp from the amdS selection marker by site-directedmutagenesis. All changes were designed to be “silent” leaving the actualprotein sequence of the amdS gene product unchanged. Removal of thesethree sites was performed simultaneously with a GeneEditor Site-DirectedMutagenesis Kit (Promega, Madison, Wis.) according to the manufacturer'sinstructions using the following primers (underlined nucleotiderepresents the changed base): AMDS3NcoMut (2050):5′-GTGCCCCATGATACGCCTCCGG-3′ (SEQ ID NO: 1) AMDS2NcoMut (2721):5′-GAGTCGTATTTCCAAGGCTCCTGACC-3′ (SEQ ID NO: 2) AMDS1NcoMut (3396):5′-GGAGGCCATGAAGTGGACCAACGG-3′ (SEQ ID NO: 3)

A plasmid comprising all three expected sequence changes was thensubmitted to site-directed mutagenesis, using a QuickChange MutagenesisKit (Stratagene, La Jolla, Calif.), to eliminate the Nco I restrictionsite at the end of the AMG terminator at position 1643. The followingprimers (underlined nucleotide represents the changed base) were usedfor mutagenesis:

Upper Primer to mutagenize the AMG terminator sequence: (SEQ ID NO: 4)5′-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACA G-3′

Lower Primer to mutagenize the AMG terminator sequence: (SEQ ID NO: 5)5′-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCT G-3′

Upper Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 6)5′-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3′

Lower Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 7)5′-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3′

Example 2 Construction of pMJ04 Expression Vector

Expression vector pMJ04 was constructed by PCR amplifying theTrichoderma reesei Cel7A cellobiohydrolase 1 terminator from Trichodermareesei RutC30 genomic DNA using primers 993429 (antisense) and 993428(sense) shown below. The antisense primer was engineered to have a Pac Isite at the 5′-end and a Spe I site at the 5′-end of the sense primer.Primer 993429 (antisense): 5′-AACGTTAATTAAGGAATCGTTTTGTGTTT-3′ (SEQ IDNO: 8) Primer 993428 (sense): 5′-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3′ (SEQID NO: 9)

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer (New England Biolabs, Beverly, Mass.), 0.3 mM dNTPs, 100ng of Trichoderma reesei RutC30 genomic DNA (which was isolated using aDNeasy Plant Maxi Kit, QIAGEN Inc., Chatsworth, Calif.), 0.3 μM primer993429, 0.3 μM primer 993428, and 2 units of Vent polymerase (NewEngland Biolabs, Beverly, Mass.). The reactions were incubated in anEppendorf Mastercycler 5333 (Hamburg, Germany) programmed as follows: 30cycles each for 30 seconds at 94° C., 30 seconds at 55° C., and 30seconds at 72° C. (15 minute final extension). The reaction productswere isolated on a 1.0% agarose gel using 40 mM Tris base-20 mM sodiumacetate-1 mM disodium EDTA (TAE) buffer where a 229 bp product band wasexcised from the gel and purified using a QIAquick Gel Extraction Kit(QIAGEN Inc., Chatsworth, Calif.) according to the manufacturer'sinstructions.

The resulting PCR fragment was digested with Pac I and Spe I and ligatedinto pAlLo1 digested with the same restriction enzymes using a RapidLigation Kit (Roche, Indianapolis, Ind.), to generate pMJ04 (FIG. 2).

Example 3 Construction of pCaHj568 Expression Vector

Expression plasmid pCaHj568 was constructed from pCaHj170 (U.S. Pat. No.5,763,254) and pMT2188. Plasmid pCaHj170 comprises the Humicola insolensendoglucanase 5 (Ce/45A) coding region. Plasmid pMT2188 was constructedas follows: The pUC19 origin of replication was PCR amplified frompCaHj483 (WO 98/00529) with primers 142779 and 142780 shown below.Primer 142780 introduces a Bbu I site in the PCR fragment. (SEQ ID NO:10) 142779: 5′-TTGAATTGAAAATAGATTGATTTAAAACTTC-3′ (SEQ ID NO: 11)142780: 5′-TTGCATGCGTAATCATGGTCATAGC-3′

The Expand PCR System (Roche Molecular Biochemicals, Basel, Switserland)was used for the amplification following the manufacturer's instructionsfor this and subsequent PCR amplifications. PCR products were separatedon an agarose gel and an 1160 bp fragment was isolated and purifiedusing a Jetquick Gel Extraction Spin Kit (Genomed, Wielandstr, Germany).

The URA3 gene was amplified from the general Saccharomyces cerevisiaecloning vector pYES2 (Invitrogen, Carlsbad, Calif.) using primers 140288and 142778 below. Primer 140288 introduces an Eco RI site in the PCRfragment. (SEQ ID NO: 12) 140288: 5′-TTGAATTCATGGGTAATAACTGATAT-3′ (SEQID NO: 13) 142778: 5′-AAATCAATCTATTTTCAATTCAATTCATCATT-3′

PCR products were separated on an agarose gel and an 1126 bp fragmentwas isolated and purified using a Jetquick Gel Extraction Spin Kit.

The two PCR fragments were fused by mixing and amplifed using primers142780 and 140288 shown above by overlap method splicing (Horton et al.,1989, Gene 77: 61-68). PCR products were separated on an agarose gel anda 2263 bp fragment was isolated and purified using a Jetquick gelextraction spin kit.

The resulting fragment was digested with EcoR I and Bbu I and ligated tothe largest fragment of pCaHj483 digested with the same enzymes. Theligation mixture was used to transform pyrF E. coli strain DB6507 (ATCC35673) made competent by the method of Mandel and Higa, 1970, J. Mol.Biol. 45: 154. Transformants were selected on solid M9 medium (Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory Press) supplemented per liter with 1 g ofcasaminoacids, 500 μg of thiamine, and 10 mg of kanamycin. A plasmidfrom one transformant was isolated and designated pCaHj527 (FIG. 3).

The NA2/tpi promoter present on pCaHj527 was subjected to site-directedmutagenesis by a simple PCR approach. Nucleotides 134-144 were convertedfrom GTACTAAAACC to CCGTTAAATTT using mutagenic primer 141223: (SEQ IDNO: 14) Primer 141223:5′-GGATGCTGTTGACTCCGGAAATTTAACGGTTTGGTCTTGCATCCC-3′

Nucleotides 423-436 were converted from ATGCAATTTAMCT to CGGCAATTTAACGGusing mutagenic primer 141222: (SEQ ID NO: 15) Primer 141222:5′-GGTATTGTCCTGCAGACGGCAATTTAACGGCTTCTGCGAATCGC-3′

The resulting plasmid was designated pMT2188 (FIG. 4).

The Humicola insolens endoglucanase 5 coding region was transferred frompCaHj170 as a Bam HI-Sal I fragment into pMT2188 digested with Bam HIand Xho I to generate pCaHj568 (FIG. 5).

Example 4 Construction of pMJ05 Expression Vector

Expression vector pMJ05 was constructed by PCR amplifying the 915 bpHumicola insolens endoglucanase 5 coding region from pCaHj568 usingprimers HiEGV-F and HiEGV-R shown below. (SEQ ID NO: 16) HiEG V-F(sense): 5′-AAGCTTAAGCATGCGTTCCTCCCCCCTCC-3′ (SEQ ID NO: 17) HiEGV-R(antisense): 5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 10 ng/μl pCaHj568 plasmid, 0.3 μM HiEGV-Fprimer, 0.3 μM HiEGV-R primer, and 2 units of Vent polymerase. Thereactions were incubated in an Eppendorf Mastercycler 5333 programmed asfollows: 5 cycles each for 30 seconds at 94° C., 30 seconds at 50° C.,and 60 seconds at 72° C., followed by 25 cycles each for 30 seconds at94° C., 30 seconds at 65° C., and 120 seconds at 72° C. (5 minute finalextension). The reaction products were isolated on a 1.0% agarose gelusing TAE buffer where a 937 bp product band was excised from the geland purified using a QIAquick Gel Extraction Kit according to themanufacturer's instructions.

This 937 bp purified fragment was used as template DNA for subsequentamplifications using the following primers: (SEQ ID NO: 18) HiEGV-R(antisense): 5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′ (SEQ ID NO: 19) HiEGV-F-overlap (sense): 5′-ACCGCGGACTGCGCATCATGCGTTCCTCCCCCCTCC-3′

Primer sequences in italics are homologous to 17 bp of the Trichodermareesei Cel7A cellobiohydrolase 1 promoter and underlined primersequences are homologous to 29 bp of the Humicola insolens endoglucanase5 coding region. The 36 bp overlap between the promoter and the codingsequence allowed precise fusion of the 994 bp fragment comprising theTrichoderma reesei Cel7A cellobiohydrolase 1 promoter to the 918 bpfragment comprising the Humicola insolens endoglucanase 5 open readingframe.

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 1 μl of the purified 937 bp PCR fragment,0.3 μM HiEGV-F-overlap primer, 0.3 μM HiEGV-R primer, and 2 units ofVent polymerase. The reactions were incubated in an EppendorfMastercycler 5333 programmed as follows: 5 cycles each for 30 seconds at94° C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120seconds at 72° C. (5 minute final extension). The reaction products wereisolated on a 1.0% agarose gel using TAE buffer where a 945 bp productband was excised from the gel and purified using a QIAquick GelExtraction Kit according to the manufacturer's instructions.

A separate PCR was performed to amplify the Trichoderma reesei Cel7Acellobiohydrolase 1 promoter sequence extending from 994 bp upstream ofthe ATG start codon of the gene from Trichoderma reesei RutC30 genomicDNA using the following primers (sense primer was engineered to have aSal I restriction site at the 5′-end): (SEQ ID NO: 20) TrCBHIpro-F(sense): 5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′ (SEQ ID NO: 21) TrCBHIpro-R(antisense): 5′-GATGCGCAGTCCGCGGT-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 100 ng/μl Trichoderma reesei RutC30genomic DNA, 0.3 μM TrCBHIpro-F primer, 0.3 μM TrCBHIpro-R primer, and 2units of Vent polymerase. The reactions were incubated in an EppendorfMastercycler 5333 programmed as follows: 30 cycles each for 30 secondsat 94° C., 30 seconds at 55° C., and 120 seconds at 72° C. (5 minutefinal extension). The reaction products were isolated on a 1.0% agarosegel using TAE buffer where a 998 bp product band was excised from thegel and purified using a QIAquick Gel Extraction Kit according to themanufacturer's instructions.

The purified 998 bp PCR fragment was used to as template DNA forsubsequent amplifications using the following primers: (SEQ ID NO: 22)TrCBHIpro-F: 5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′ (SEQ ID NO: 23)TrCBHIpro-R-overiap: 5′-GGAGGGGGGAGGAACGCAT GATGCGCAGTCCGCGGT-3′

Sequences in italics are homologous to the 17 bp of the Trichodermareesei cbh1 promoter and underlined sequences are homologous to the 29bp of the Humicola insolens endoglucanase 5 coding region. The 36 bpoverlap between the promoter and the coding sequence allowed precisefusion of the 994 bp fragment comprising the Trichoderma reesei Cel7Apromoter to the 918 bp fragment comprising the Humicola insolensendoglucanase 5 open reading frame.

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 1 μl of the purified 998 bp PCR fragment,0.3 μM TrCBH1pro-F primer, 0.3 μM TrCBH1pro-R-overlap primer, and 2units of Vent polymerase. The reactions were incubated in an EppendorfMastercycler 5333 programmed as follows: 5 cycles each for 30 seconds at94° C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120seconds at 72° C. (5 minute final extension). The reaction products wereisolated on a 1.0% agarose gel using TAE buffer where a 1017 bp productband was excised from the gel and purified using a QIAquick GelExtraction Kit according to the manufacturer's instructions.

The 1017 bp Trichoderma reesei Cel7A cellobiohydrolase 1 promoter PCRfragment and the 945 bp Humicola insolens endoglucanase 5 PCR fragmentwere used as template DNA for subsequent amplification using thefollowing primers to precisely fuse the 994 bp Trichoderma reesei Cel7Acellobiohydrolase 1 promoter to the 918 bp Humicola insolensendoglucanase 5 coding region using overlapping PCR. (SEQ ID NO: 24)TrCBHIpro-F: 5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′ (SEQ ID NO: 25)HiEGV-R: 5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 0.3 μM TrCBH1pro-F primer, 0.3 μM HiEGV-Rprimer, and 2 units of Vent polymerase. The reactions were incubated inan Eppendorf Mastercycler 5333 programmed as follows: 5 cycles each for30 seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C.,followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65°C., and 120 seconds at 72° C. (5 minute final extension). The reactionproducts were isolated on a 1.0% agarose gel using TAE buffer where a1926 bp product band was excised from the gel and purified using aQIAquick Gel Extraction Kit according to the manufacturer'sinstructions.

The resulting 1926 bp fragment was cloned into pCR-Blunt-II-TOPO vector(Invitrogen, Carlsbad, Calif.) using a ZeroBlunt TOPO PCR Cloning Kitfollowing the manufacturer's protocol. The resulting plasmid wasdigested with Not I and Sal I and the 1926 bp fragment purified andligated into pMJ04, which was also digested with the same tworestriction enzymes, to generate pMJ05 (FIG. 6).

Example 5 Construction of pSMai135

The Aspergillus oryzae beta-glucosidase coding region (WO 2002/095014,E. coli DSM 14240, minus the putative signal sequence, see FIG. 7, SEQID NOs: 26 and 27) from Lys-20 to the TAA stop codon was PCR amplifiedfrom pJaL660 (WO 2002/095014) as template with primer 993728 (sense) andprimer 993727 (antisense) shown below. Sequences in italics arehomologous to 20 bp of the Humicola insolens endoglucanase 5 signalsequence and sequences underlined are homologous to 22 bp of theAspergillus oryzae beta-glucosidase coding region. A Spe I site wasengineered into the 5′ end of the antisense primer. (SEQ ID NO: 28)Primer 993728: 5′-TGCCGGTGTTGGCCCTTGCC AAGGATGATCTCGCGTACTCCC-3′ (SEQ IDNO: 29) Primer 993727: 5′-GACTAGTCTTACTGGGCCTTAGGCAGCG-3′

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 10 ng/μl pJaL660, 6.4 μM primer 993728, 3.2 μMprimer 993727, 1 mM MgCl₂, and 2.5 units of Pfx polymerase. Thereactions were incubated in an Eppendorf Mastercycler 5333 programmed asfollows: 30 cycles each for 60 seconds at 94° C., 60 seconds at 55° C.,and 180 seconds at 72° C. (15 minute final extension). The reactionproducts were isolated on a 1.0% agarose gel using TAE buffer where a2523 bp product band was excised from the gel and purified using aQIAquick Gel Extraction Kit according to the manufacturer'sinstructions.

A separate PCR amplification was performed to amplify 1000 bp of theTrichoderma reesei Cel7A cellobiohydrolase 1 promoter and 63 bp of theputative Humicola insolens endoglucanase 5 signal sequence (ATG startcodon to Ala-21, FIG. 8, SEQ ID NOs: 30 (DNA sequence) and 31 (deducedamino acid sequence); accession no. AAB03660 for DNA sequence), usingprimer 993724 (sense) and primer 993729 (antisense) shown below. Primersequences in italics are homologous to 20 bp of the Humicola insolensendoglucanase 5 signal sequence and underlined primer sequences arehomologous to 22 bp of the Aspergillus oryzae beta-glucosidase codingregion. Plasmid pMJ05, which comprises the Humicola insolensendoglucanase 5 coding region under the control of the Trichodermareesei Cel7A cellobiohydrolase 1 promoter, was used as a template togenerate a 1063 bp fragment comprising the Trichoderma reesei Cel7Acellobiohydrolase 1 promoter/Humicola insolens endoglucanase 5 signalsequence fragment. A 42 bp of overlap was shared between the Trichodermareesei Cel7A promoter/Humicola insolens endoglucanase 5 signal sequenceand the Aspergillus oryzae coding sequence to provide a perfect linkagebetween the promoter and the ATG start codon of the 2523 bp Aspergillusoryzae beta-glucosidase. (SEQ ID NO: 32) Primer 993724:5′-ACGCGTCGACCGAATGTAGGATTGTTATCC-3′ (SEQ ID NO: 33) Primer 993729:5′-GGGAGTACGCGAGATCATCCTT GGCAAGGGCCAACACCGGCA-3′

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 10 ng/μl pMJ05, 6.4 μM primer 993728, 3.2 μMprimer 993727, 1 mM MgCl₂, and 2.5 units of Pfx polymerase. Thereactions were incubated in an Eppendorf Mastercycler 5333 programmed asfollows: 30 cycles each for 60 seconds at 94° C., 60 seconds at 60° C.,and 240 seconds at 72° C. (15 minute final extension). The reactionproducts were isolated on a 1.0% agarose gel using TAE buffer where a1063 bp product band was excised from the gel and purified using aQIAquick Gel Extraction Kit according to the manufacturer'sinstructions.

The purified overlapping fragments were used as a template foramplification using primer 993724 (sense) and primer 993727 (antisense)described above to precisely fuse the 1063 bp Trichoderma reesei Cel7Acellobiohydrolase 1 promoter/Humicola insolens endoglucanase V5signalsequence fragment to the 2523 bp of Aspergillus oryzae beta-glucosidaseby overlapping PCR.

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 6.4 μM primer 993724, 3.2 μM primer 993727, 1 mMMgCl₂, and 2.5 units of Pfx polymerase. The reactions were incubated inan Eppendorf Mastercycler 5333 programmed as follows: 30 cycles each for60 seconds at 94° C., 60 seconds at 60° C., and 240 seconds at 72° C.(15 minute final extension). The reaction products were isolated on a1.0% agarose gel using TAE buffer where a 3591 bp product band wasexcised from the gel and purified using a QIAquick Gel Extraction Kitaccording to the manufacturer's instructions.

The resulting 3591 bp fragment was digested with Sal I and Spe I andligated into pMJ04 digested with the same restriction enzymes togenerate pSMai135 (FIG. 9).

Example 6 Expression of Aspergillus oryzae Beta-Glucosidase inTrichoderma reesei

Plasmid pSMai135 encoding the mature Aspergillus oryzae beta-glucosidaseenzyme, linked to Humicola insolens endoglucanase 5 secretion signal(FIG. 8, SEQ ID NOs: 30 (DNA sequence) and 31 (deduced amino acidsequence), was introduced into Trichoderma reesei RutC30 by PEG-mediatedtransformation. The expression plasmid contains the Aspergillus nidulansamdS gene to enable transformants to grow on acetamide as the solenitrogen source.

Protoplast preparation and transformation was performed using a modifiedprotocol by Penttila et al., 1987, Gene 61: 155-164. Briefly,Trichoderma reesei RutC30 was cultivated at 27° C. and 90 rpm in 25 mlof YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17hours. Mycelia was collected by filtration using Millipore's VacuumDriven Disposable Filtration System (Millipore, Bedford, Mass.) andwashed twice with deionized water and twice with 1.2 M sorbitol.Protoplasts were generated by suspending the washed mycelia in 20 ml of1.2 M sorbitol containing 15 mg of Glucanex (Novozymes A/S, Bagsvaerd,Denmark) per ml and 0.36 units of chitinase (Sigma Chemical Co., St.Louis, Mo.) per ml and incubating for 15-25 minutes at 34° C. withgentle shaking at 90 rpm. Protoplasts were collected by centrifuging for7 minutes at 400×g and washed twice with cold 1.2 M sorbitol. Theprotoplasts were counted using a haemacytometer and re-suspended in STCto a final concentration of 1×10⁸ protoplasts per ml. Excess protoplastswere stored in a Cryo 1° C. Freezing Container (Nalgene, Rochester,N.Y.) at −80° C.

Approximately 7 μg of pSMai135 digested with Pme I was added to 100 μlof protoplast solution and mixed gently, followed by 260 μl of PEGbuffer, mixed, and incubated at room temperature for 30 minutes. STC (3ml) was then added, mixed and the transformation solution was platedonto COVE plates using Aspergillus nidulans amdS selection. The plateswere incubated at 28° C. for 5-7 days. Transformants were sub-culturedonto COVE2 plates and grown at 28° C.

Sixty-seven transformants (SMA135-01 to SMA135-67) harboring theAspergillus oryzae beta-glucosidase gene were randomly selected andcultured in 125 ml baffled shake flasks containing 25 ml ofcellulase-inducing media at 28° C. and 200 rpm for 7 days. Trichodermareesei RutC30 was run as a control. Culture broth samples were removed 7days post-inoculation, centrifuged at 15,700×g for 5 minutes in amicro-centrifuge, and the supernatants transferred to new tubes. Sampleswere stored at 4° C. until enzyme assay. The supernatants were assayedfor beta-glucosidase activity using p-nitrophenyl-beta-D-glucopyranosideas substrate, as described above.

A number of SMA135 transformants showed beta-glucosidase activitiesseveral fold more than that of Trichoderma reesei RutC30. TransformantSMA135-04 produced the highest beta-glucosidase activity having 7 timesmore beta-glucosidase activity than produced by Trichoderma reeseiRutC30 as a control.

SDS polyacrylamide electrophoresis was carried out using CriterionTris-HCl (5% resolving) gels (BioRad, Hercules, Calif.) with TheCriterion System (BioRad, Hercules, Calif.). Five μl of day 7supernatants (see above) were suspended in 2× concentration of LaemmliSample Buffer (BioRad, Hercules, Calif.) and boiled for 3 minutes in thepresence of 5% beta-mercaptoethanol. The supernatant samples were loadedonto a polyacrylamide gel and subjected to electrophoresis with 1×Tris/Glycine/SDS as running buffer (BioRad, Hercules, Calif.). Theresulting gel was stained with BioRad's Bio-Safe Coomassie Stain.

Twenty-six of the 67 Trichoderma reesei SMA135 transformants produced aprotein of approximately 110 kDa that was not visible in the hoststrain, Trichoderma reesei RutC30. Trichoderma reesei transformant,SMA135-04, produced the highest level of beta-glucosidase.

Example 7 Production of Recombinant Aspergillus oryzae Beta-Glucosidasein Trichoderma reesei by Fermentation

Fermentation was performed on Trichoderma reesei strain SMA135-04 toproduce quantities of Aspergillus oryzae beta-glucosidase. Trichodermareesei RutC30 (host strain) was run as a control. Fermentations wereperformed using the standard conditions as described by Mandels andWeber, 1969, Adv. Chem. Ser. 95: 391-413) on 2% cellulose. Thefermentations ran for 165 hours at which time the final fermentationbroths were centrifuged and the supernatants stored at −20° C. untilbeta-glucosidase activity assay using the procedure described earlier.

Beta-glucosidase activity on the Trichoderma reesei SMA135-04fermentation sample was determined to be approximately 8 times moreactive than that of Trichoderma reesei RutC30.

Example 8 Identification of a Beta-Glucosidase Family GH3A Gene in theGenomic Sequence of Aspergillus fumigatus

A Blast search (Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402)of the Aspergillus fumigatus partial genome sequence (The Institute forGenomic Research, Rockville, Md.) was carried out using as query abeta-glucosidase protein sequence from Aspergillus aculeatus (AccessionNo. P48825). Several genes were identified as putative Family GH3Ahomologs based upon a high degree of similarity to the query sequence atthe amino acid level. One genomic region of approximately 3000 bp withgreater than 70% identity to the query sequence at the amino acid levelwas chosen for further study.

Example 9 Aspergillus fumigatus Genomic DNA Extraction

Aspergillus fumigatus was grown in 250 ml of potato dextrose medium in abaffled shake flask at 37° C. and 240 rpm. Mycelia were harvested byfiltration, washed twice in TE (10 mM Tris-1 mM EDTA), and frozen underliquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to afine powder, which was resuspended in pH 8.0 buffer containing 10 mMTris, 100 mM EDTA, 1% Triton X-100, 0.5 M guanidine-HCl, and 200 mMNaCl. DNase free RNase A was added at a concentration of 20 mg/liter andthe lysate was incubated at 37° C. for 30 minutes. Cellular debris wasremoved by centrifugation, and DNA was isolated by using a Qiagen Maxi500 column (QIAGEN Inc., Chatsworth, Calif.). The columns wereequilibrated in 10 ml of QBT washed with 30 ml of QC, and eluted with 15ml of QF (all buffers from QIAGEN Inc., Chatsworth, Calif.). DNA wasprecipitated in isopropanol, washed in 70% ethanol, and recovered bycentrifugation. The DNA was resuspended in TE buffer.

Example 10 Construction of pAILo2 Expression Vector

The amdS gene of pAILo1 (Example 1) was swapped with the Aspergillusnidulans pyrG gene. Plasmid pBANe10 (FIG. 11) was used as a source forthe pyrg gene. Analysis of the sequence of pBANe10 showed that the pyrGmarker was contained within an Nsi I restriction fragment and does notcontain either Nco I or Pac I restriction sites. Since the amdS is alsoflanked by Nsi I restriction sites the strategy to switch the selectionmarker was a simple swap of Nsi I restriction fragments. Plasmid DNAfrom pAlLo1 and pBANe10 were digested with the restriction enzyme Nsi Iand the products purified by agarose gel electrophoresis using standardprocedures. The Nsi I fragment from pBANe10 containing the pyrG gene wasligated to the backbone of pAlLo1 to replace the original Nsi I DNAfragment containing the amdS gene. Recombinant clones were analyzed byrestriction digest to determine whether they had the correct insert andcorrect orientation. A clone with the pyrg gene transcribed in thecounterclockwise direction was selected. The new plasmid was designatedpAlLo2 (FIG. 12).

Example 11 Cloning of the Family GH3A Beta-Glucosidase Gene andConstruction of an Aspergillus oryzae Expression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a Aspergillus fumigatus gene encoding a Family GH3Abeta-glucosidase from the genomic DNA prepared in Example 9. An InFusionCloning Kit (BD Biosciences, Palo Alto, Calif.) was used to clone thefragment directly into the expression vector, pAlLo2, without the needfor restriction digests and ligation. (SEQ ID NO: 34) Forward primer:5′-ACTGGATTTACCATGAGATTCGGTTGGCTCG-3′ (SEQ ID NO: 35) Reverse primer:5′-AGTCACCTCTAGTTACTAGTAGACACGGGGC-3′

Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 100 ng of Aspergillus fumigatus genomic DNA, 1× PfxAmplification Buffer (Invitrogen, Carlsbad, Calif.), 1.5 μl of 10 mMblend of dATP, dTTP, dGTP, and dCTP, 2.5 units of Platinum Pfx DNAPolymerase (Invitrogen, Carlsbad, Calif.), 1 μl of 50 mM MgSO₄ and 2.5μl of 10× pCRx Enhancer solution (Invitrogen, Carlsbad, Calif.) in afinal volume of 50 μl. The amplification conditions were one cycle at94° C. for 2 minutes; 30 cycles each at 94° C. for 15 seconds, 55° C.for 30 seconds, and 68° C. for 3 minutes. The heat block then went to a4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 3 kb product band was excised from the gel and purifiedusing a QIAquick Gel Extraction Kit according to the manufacturer'sinstructions.

The fragment was then cloned into the pAlLo2 expression vector using anInfusion Cloning Kit. The vector was digested with restrictionendonucleases Nco I and Pac I (using conditions specified by themanufacturer). The fragment was purified by gel electrophoresis andQIAquick Gel Extraction Kit. The gene fragment and digested vector wereligated together in a reaction resulting in the expression plasmidpEJG97 (FIG. 13) in which transcription of the Family GH3Abeta-glucosidase gene was under the control of the NA2-tpi promoter. Theligation reaction (50 μl) was composed of 1× InFusion Buffer (BDBiosciences, Palo Alto, Calif.), 1×BSA (BD Biosciences, Palo Alto,Calif.), 1 μl of Infusion enzyme (diluted 1:10) (BD Biosciences, PaloAlto, Calif.), 150 ng of pAlLo2 digested with Nco I and Pac 1, and 50 ngof the Aspergillus fumigatus beta-glucosidase purified PCR product. Thereaction was incubated at room temperature for 30 minutes. One μl of thereaction was used to transform E. coli XL10 Solopac Gold cells(Stratagene, La Jolla, Calif.). An E. coli transformant containing thepEJG97 plasmid was detected by restriction digestion and plasmid DNA wasprepared using a BioRobot 9600 (QIAGEN Inc., Chatsworth, Calif.)

Example 12 Characterization of the Aspergillus fumigatus GenomicSequence Encoding a Family GH3A Beta-Glucosidase

DNA sequencing of the Aspergillus fumigatus beta-glucosidase gene frompEJG97 was performed with a Perkin-Elmer Applied Biosystems Model 377 XLAutomated DNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., FosterCity, Calif.) using dye-terminator chemistry (Giesecke et al., 1992,Journal of Virology Methods 38: 47-60) and primer walking strategy.Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash.).

A gene model for the Aspergillus fumigatus sequence was constructedbased on similarity to homologous genes from Aspergillus aculeatus,Aspergillus niger, and Aspergillus kawachii. The nucleotide sequence(SEQ ID NO: 36) and deduced amino acid sequence (SEQ ID NO: 37) areshown in FIG. 10. The genomic fragment encodes a polypeptide of 863amino acids, interrupted by 8 introns of 62, 55, 58, 63, 58, 58, 63 and51 bp. The % G+C content of the gene is 54.3%. Using the SignalPsoftware program (Nielsen et al., 1997, Protein Engineering 10: 1-6), asignal peptide of 19 residues was predicted. The predicted matureprotein contains 844 amino acids with a molecular mass of 91.7 kDa.

A comparative alignment of beta-glucosidase sequences was determinedusing the Clustal W method (Higgins, 1989, CABIOS 5: 151-153) using theLASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with anidentity table and the following multiple alignment parameters: Gappenalty of 10 and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, windows=5, and diagonals=5. Thealignment showed that the deduced amino acid sequence of the Aspergillusfumigatus beta-glucosidase gene shared 78%, 76%, and 76% identity to thededuced amino acid sequences of the Aspergillus aculeatus (accessionnumber P48825), Aspergillus niger (accession number 000089), andAspergillus kawachii (accession number P87076) beta-glucosidases.

Example 13 Expression of the Aspergillus fumigatus Family GH3ABeta-Glucosidase Gene in Aspergillus oryzae JaL250

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Five μgof pEJG97 (as well as pAlLo2 as a vector control) was used to transformAspergillus oryzae JaL250.

The transformation of Aspergillus oryzae JaL250 with pEJG97 yieldedabout 100 transformants. Ten transformants were isolated to individualPDA plates.

Confluent PDA plates of five of the ten transformants were washed with 5ml of 0.01% Tween 20 and inoculated separately into 25 ml of MDU2BPmedium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm.Five days after incubation, 0.5 μl of supernatant from each culture wasanalyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad,Calif.) according to the manufacturer's instructions. SDS-PAGE profilesof the cultures showed that one of the transformants (designatedtransformant 1) had a major band of approximately 130 kDa.

A confluent plate of transformant 1 (grown on PDA) was washed with 10 mlof 0.01% Tween 20 and inoculated into a 2 liter Fernbach containing 400ml of MDU2BP medium to generate broth for characterization of theenzyme. The flask was harvested on day 5 and filtered using a 0.22 μm GPExpress plus Membrane (Millipore, Bedford, Mass.).

Prior to hydrolysis experiments, Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae) was desalted andexchanged to 50 mM sodium acetate pH 5.0 buffer, using a CentriconPlus-20 centrifugal filter with Biomax-5 membrane (5000 NMWL; Millipore,Bedford, Mass.).

Example 14 Construction of pMJ09

Vector pMJ06 was constructed by PCR amplifying the Trichoderma reeseiCel7A cellobiohydrolase 1 gene (cbh1) promoter from Trichoderma reeseiRutC30 genomic DNA using primers 993696 (antisense) and 993695 (sense)shown below. The antisense primer was engineered to have a Sal I site atthe 5′-end of the sense primer and an Nco I site at the 5′-end of theantisense primer. (SEQ ID NO: 38) Primer 993695 (sense):5′-ACTAGTCGACCGAATGTAGGATTGTT-3′ (SEQ ID NO: 39) Primer 993696(antisense): 5′-TGACCATGGTGCGCAGTCC-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 100 ng Trichoderma reesei RutC30 genomicDNA (which was prepared using a QIAGEN DNeasy Plant Maxi Kit), 0.3 μMprimer 993696, 0.3 μM primer 993695, and 2 units of Vent polymerase. Thereactions were incubated in an Eppendorf Mastercycler 5333 programmed asfollows: 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C.,and 60 seconds at 72° C. (15 minute final extension).

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 988 bp product band was excised from the gel and purifiedusing a QIAGEN QIAquick Gel Extraction Kit according to themanufacturer's instructions.

The resulting PCR fragment was digested with Nco I and Sal I and ligatedinto pMJ04 digested with the same restriction enzymes, using a RapidLigation Kit, to generate pMJ06 (FIG. 14).

Expression vector pMJ09 was constructed by PCR amplifying theTrichoderma reesei Cel7A cellobiohydrolase 1 gene (cbh1) terminator fromTrichoderma reesei RutC30 genomic DNA using primers 993843 (antisense)and 99344 (sense) shown below. The antisense primer was engineered tohave a Pac I and a Spe I sites at the 5′-end and a Pvu I site at the5′-end of the sense primer. (SEQ ID NO: 40) Primer 993844 (sense):5′-CGATCGTCTCCCTATGGGTCATTACC-3′ (SEQ ID NO: 41) Primer 993843(antisense): 5′-ACTAGTTAATTAAGCTCCGTGGCGAAAG-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30genomic DNA (which was extracted using a QIAGEN DNeasy Plant Maxi Kit),0.3 μM primer 993844, 0.3 μM primer 993843, and 2 units of Ventpolymerase The reactions were incubated in an Eppendorf Mastercycler5333 programmed as follows: 30 cycles each for 30 seconds at 94° C., 30seconds at 55° C., and 60 seconds at 72° C. (15 minute final extension).

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 473 bp product band was excised from the gel and purifiedusing a QIAGEN QIAquick Gel Extraction Kit according to themanufacturer's instructions.

The resulting PCR fragment was digested with Pvu I and Spe I and ligatedinto pMJ06, digested with Pac I and Spe I using a Rapid Ligation Kit, togenerate pMJ09 (FIG. 15).

Example 15 Cloning, Characterization, and Expression of the Family 7Trichoderma reesei Endoglucanse (eg1) Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a Trichoderma reesei gene encoding a putative Family 7endoglucanase from cDNA prepared from Trichoderma reesei RutC30 by usinga Cells-to-cDNA Kit (Ambion, Austin, Tex.) according to themanufacturer's instructions. Forward primer: (SEQ ID NO: 42)5′-CTTCACCATGGCGCCCTCAGTTACACTGC-3′ Reverse primer: (SEQ ID NO: 43)5′-GCCGTTAATTAAGGCAAGTCAACGCTCTAAAGG-3′

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 100 ng of Trichoderma reesei cDNA, 1× Pwo AmplificationBuffer (Roche, Indianapolis, Ind.), 4 μl of 10 mM blend of dATP, dTTP,dGTP, and dCTP, and 2.5 units of Pwo DNA Polymerase (Roche,Indianapolis, Ind.) in a final volume of 50 μl. The amplificationconditions were 1 cycle at 94° C. for 2 minutes; 35 cycles each at 94°C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes. Theheat block then went to a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 1.5 kb product band was excised from the gel and purifiedusing a QIAquick Gel Extraction Kit (QIAGEN Inc., Chatsworth, Calif.)according to the manufacturer's instructions.

The fragment was then cloned into pAlLo2. Both fragment and vector weredigested with Nco I and Pac I, and then were purified by gelelectrophoresis and Qiaquick gel purification. The digested genefragment and vector were ligated together in a reaction resulting in theexpression plasmid pJZHEG1 (FIG. 16) in which transcription of the eg1gene was under the control of the TAKA promoter. The ligation reaction(40 μl) was composed of 1× ligation Buffer (Roche, Indianapolis, Ind.),1×DNA dilution Buffer (Roche, Indianapolis, Ind.), 5.0 units of T4 DNAligase enzyme (Roche, Indianapolis, Ind.), 100 ng of pAILo2 digestedwith Nco I and Pac I, and 100 ng of the Trichoderma reesei eg1 PCRproduct. The reaction was incubated at room temperature for 30 minutes.One μl of the reaction was used to transform E. coli XL10 Solopac Goldcells (Stratagene, La Jolla, Calif.). An E. coli transformant containingthe pJZHEG1 plasmid was detected by restriction digestion and plasmidDNA was prepared using a BioRobot 9600 (QIAGEN Inc., Chatsworth,Calif.).

DNA sequencing of the Trichoderma reesei eg1 from pJZHEG1 was performedwith a Perkin-Elmer Applied Biosystems Model 377 XL Automated DNASequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.)using dye-terminator chemistry according to the manufacturer'sinstructions. Nucleotide sequence data were scrutinized for quality andall sequences were compared to each other with assistance of PHRED/PHRAPsoftware (University of Washington, Seattle, Wash.). A comparativealignment of eg1 sequence was determined using the Clustal W method(Higgins, 1989, CABIOS 5: 151-153) with the Vector NTI software(InfoMax, San Francisco, Calif.). The alignment indicated that thededuced amino acid sequence of the Trichoderma reesei eg1 gene isidentical to the deduced amino acid sequence of the Trichoderma reeseiEG1 (accession number AAA34212, Penttila et al., 1986, Gene 45:253-263).

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. Five μg of pJZHEG1 (as wellas pAILo2 as a vector control) was used to transform Aspergillus oryzaeJaL250.

The transformation of Aspergillus oryzae JaL250 with pJZHEG1 yieldedabout 40 transformants per μg DNA. Twenty transformants were isolated toindividual PDA plates. Each plate was washed with 5 ml of 0.01% Tween 20and inoculated separately into 25 ml of MDU2BP medium in 125 ml glassshake flasks and incubated at 34° C., 250 rpm. Five days afterincubation, 0.5 μl of supernatant from each culture was analyzed using8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions. SDS-PAGE profiles of thecultures showed that one of the transformants (designated transformant9) had a major band of approximately 58 kDa.

A confluent plate of transformant 9 (grown on PDA) was washed with 10 mlof 0.01% Tween 20 and inoculated into a 2 liter Fernbach containing 400ml of MDU2BP medium to generate broth for characterization of theenzyme. The flask was harvested on day 5 and filtered using a 0.22 μm GPExpress plus Membrane (Millipore, Bedford, Mass.). Endoglucanaseactivity was determined as described herein.

Prior to hydrolysis experiments, Trichoderma reesei endoglucanase I(recombinantly produced in Aspergillus oryzae) was desalted andexchanged into 50 mM sodium acetate pH 5.0 buffer using a CentriconPlus-20 centrifugal filter with Biomax-5 membrane (5000 NMWL; Millipore,Bedford, Mass.).

Example 16 Preparation of Pretreated Corn Stover

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using dilute sulfuric acid. Thefollowing conditions were used for the pretreatment: 1.4 wt % sulfuricacid at 165° C. and 107 psi for 8 minutes. The water-insoluble solids inthe pretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003. Prior to enzymatic hydrolysis, the PCS was washed with a largevolume of DDI water on a glass filter. The dry weight of thewater-washed PCS was found to be 24.54%.

Example 17 Preparation of Lignacious Residue

Lignacious residue was prepared by incubating 5% PCS with cell-freefiltered fermentation broth of Aspergillus oryzae beta-glucosidase(recombinantly produced in Trichoderma reesei) at 10 mg per g of PCSunder constant stirring in a 900-ml reaction for 6 days at pH 5.0, 50°C. After hydrolysis, liquid hydrolysate was removed by centrifugation at3836×g for 10 minutes, and the solid lignin-enriched residue was washedfour times with 250 ml of deionized water. The residue was thenincubated for 5 days in 250 ml of deionized water under constantstirring at 65° C. to inactivate any remaining adsorbed enzyme. Afteradditional washing with deionized water followed by centrifugation asdescribed above, approximately 100 ml of a 15% suspension of lignaceousresidue in deionized water was obtained.

Example 18 Sugar Analysis

Twenty μl aliquots were removed from PCS hydrolysis reactions atspecified time points using an 8-channel pipettor, and added to 180 μlof alkaline mixture (102 mM Na₂CO₃ plus 58 mM NaHCO₃) in a MultiScreenHV 96-well filtration plate (Millipore, Bedford, Mass.) to terminate thereaction. The samples were vacuum-filtered into another flat-bottomedmicroplate to remove the PCS residue. After appropriate dilution, thefiltrates were analyzed for reducing sugar (RS) using p-hydroxybenzoicacid hydrazide (PHBAH) assay (Lever M., 1973, Colorimetric andfluorometric carbohydrate determination with p-hydroxybenzoic acidhydrazide, Biochemical Medicine 7: 274-281) in a microplate format.

A 90-μl aliquot of the diluted sample was placed into each well of a96-well conical-bottomed microplate (Corning Inc., Acton, Mass., Costar,clear polycarbonate). The assay was started by adding 60 μl of 1.25%PHBAH in 2% sodium hydroxide to each well. The uncovered plate washeated on a custom-made heating block for 10 minutes at 95° C. After themicroplate was cooled to room temperature, 35 μl of deionized water wasadded to each well. A 100-μl aliquot was removed from each well andtransferred to a flat-bottomed 96-well plate (Corning Inc., Acton,Mass., Costar, medium binding polystyrene). The absorbance at 410 nm(A₄₁₀) was measured using a SpectraMAX Microplate Reader (MolecularDevices, Sunnyvale, Calif.). The A₄₁₀ value was translated into glucoseequivalents using a standard curve.

The standard curve was obtained with six glucose standards (0.005,0.010, 0.025, 0.050, 0.075, and 0.100 mg/ml), which were treatedsimilarly to the samples. Glucose standards were prepared by diluting 10mg/ml stock glucose solution with deionized water.

The degree of cellulose conversion to reducing sugar (hydrolysis yield,%) was calculated using the following equation: $\begin{matrix}{{{RS}\quad{Yield}_{(\%)}} = {{RS}_{({{mg}/{ml}})}^{*}100^{*}{162/\left( {{Cellulose}_{({{mg}/{ml}})}^{*}180} \right)}}} \\{= {{RS}_{({{mg}/{ml}})}^{*}{100/\left( {{Cellulose}_{({{mg}/{ml}})}^{*}1.111} \right)}}}\end{matrix}$In this equation, RS is the concentration of reducing sugar in solutionmeasured in glucose equivalents (mg/ml), and the factor 1.111 reflectsthe weight gain in converting cellulose to glucose.

Example 19 Enhanced Production of Sugars from PCS Using Softanol® 90Non-Ionic Surfactant at 50° C.

Hydrolysis of PCS (5% w/v on a dry weight basis) by Celluclast 1.5L(2.5, 5, 10, and 20 mg/g PCS) supplemented with desalted and bufferexchanged Aspergillus oryzae beta-glucosidase (recombinantly produced inAspergillus oryzae) at 0.6 mg per g of PCS was carried out in 50 mMsodium acetate pH 5.0 in a 10-ml volume with intermittent stirring at50° C. Samples were taken at different time-points and analyzed forreducing sugars as described in Example 18. Reactions containingCelluclast 1.5L at 2.5 and 5 mg per g of PCS were supplemented with 0.5%v/v Softanol® 90 (0.1 ml/g PCS), and the results were compared withcontrol reactions without the surfactant.

The results as shown in Table 2 and FIG. 17 demonstrated that additionof Softanols 90 increased the hydrolysis yield of reducing sugars afterincubation for 125 hours from 70% to 86% for reactions with 2.5 mgCelluclast/g PCS (23% improvement), and from 83% to 94% for reactionswith 5 mg Celluclast/g PCS (14% improvement). Using Softanol® 90, it waspossible to reduce the enzyme loading by a factor of two compared toreactions without the surfactant, while achieving the same 125 hourhydrolysis yield. TABLE 2 Effect of Softanol ® 90 and Lutensol ® AT80(0.1 ml/g PCS) on Hydrolysis of PCS (5%) by Celluclast 1.5 L at 50° C.Sur- Sur- factant, 125-hour Improve- factant, ml/g Conver- ment atReaction % PCS sion, % 125 h, % Celluclast (2.5 mg/g PCS) 0.0 0.0 70 NACelluclast (5 mg/g PCS) 0.0 0.0 83 NA Celluclast (10 mg/g PCS) 0.0 0.097 NA Celluclast (20 mg/g PCS) 0.0 0.0 96 NA Celluclast (2.5 mg/g PCS) +0.5 0.1 86 23 Softanol ® 90 (0.1 ml/g PCS) Celluclast (5 mg/g PCS) + 0.50.1 94 14 Softanol ® 90 (0.1 ml/g PCS) Celluclast (2.5 mg/g PCS) + 0.50.1 82 17 Lutensol ® AT80 (0.1 ml/g PCS) Celluclast (5 mg/g PCS) + 0.50.1 90  9 Lutensol ® AT80 (0.1 ml/g PCS)

Example 20 Enhanced Production of Sugars from PCS using Lutensol® AT80Non-Ionic Surfactant at 50° C.

Example 19 was repeated, except that Lutensol® AT80 was used at 0.1 ml/gPCS (0.5% v/v).

The results as shown in Table 2 and FIG. 18 demonstrated that additionof Lutensol® AT80 increased the 125-hour hydrolysis yield of reducingsugars from 70% to 82% for reactions with 2.5 mg Celluclast/g PCS (17%improvement), and from 83% to 90% for reactions with 5 mg Celluclast/gPCS (9% improvement).

Example 21 Enhanced Production of Sugars from PCS using Softanol® 90 at50° C. and 55° C.

Example 19 was repeated, except that additional loading of Celluclast1.5L was included (1.25 mg/g PCS), Softanol® 90 was used at 0.2 ml/g PCS(1.0% v/v), and the hydrolysis was run at two temperatures, 50° C. and55° C. Softanol® 90 was added to the reactions containing 1.25, 2.5, and5 mg of Celluclast 1.5L per g of PCS, and the results were compared withcontrol reactions without the surfactant. The results are shown in Table3 and FIGS. 19 and 20.

At 50° C., addition of Softanol® 90 increased the 120-hour hydrolysisyield of reducing sugars by 14-20% compared to reactions without thesurfactant. FIG. 19 shows that in the presence of Softanol® 90, theenzyme loading could be reduced by a factor of two compared to reactionswithout the surfactant while maintaining the same hydrolysis yield.

At 55° C., Softanol® 90 showed more significant improvement than at 50°C. Addition of Softanol® 90 at 55° C. increased the 120-hour hydrolysisyields of reducing sugars by 36-59% compared to reactions without thesurfactant. FIG. 20 shows that in the presence of Softanol® 90, fourtimes lower enzyme loadings compared to reactions without the surfactantcould achieve comparable hydrolysis yields of reducing sugars. TABLE 3Effect of Softanol ® 90 (0.2 ml/g PCS) on Hydrolysis of PCS (5%) byCelluclast 1.5 L at 50° C. and 55° C. Softanol ® 90, ml/g 120-hourImprovement T° C. Reaction PCS Conversion % at 120 h, % 50 Celluclast(1.25 mg/g PCS) 0.0 52 NA Celluclast (2.5 mg/g PCS) 0.0 64 NA Celluclast(5 mg/g PCS) 0.0 80 NA Celluclast (10 mg/g PCS) 0.0 92 NA Celluclast (20mg/g PCS) 0.0 98 NA Celluclast (1.25 mg/g PCS) + 0.2 62 20 Softanol ® 90Celluclast (2.5 mg/g PCS) + 0.2 75 17 Softanol ® 90 Celluclast (5 mg/gPCS) + 0.2 91 14 Softanol ® 90 55 Celluclast (1.25 mg/g PCS) 0.0 36 NACelluclast (2.5 mg/g PCS) 0.0 42 NA Celluclast (5 mg/g PCS) 0.0 60 NACelluclast (10 mg/g PCS) 0.0 78 NA Celluclast (20 mg/g PCS) 0.0 83 NACelluclast (1.25 mg/g PCS) + 0.2 57 59 Softanol ® 90 Celluclast (2.5mg/g PCS) + 0.2 65 54 Softanol ® 90 Celluclast (5 mg/g PCS) + 0.2 82 36Softanol ® 90

Example 22 Softanol® 90 Dose Dependence for Hydrolysis of PCS byCelluclast 1.5L at 50° C.

Example 19 was repeated, except that Softanol® 90 was added at 0.05,0.1, 0.2, 0.3, and 0.4 ml/g PCS to reactions with 2 mg of Celluclast1.5L per g of PCS, and desalted and buffer exchanged Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae) wasadded at 3% of Celluclast loading (0.6 mg/g PCS).

The results are shown in Table 4 and FIG. 21. The optimal loading ofSoftanol® 90 was found to be 0.1 ml per g of PCS, which corresponded toa Softanol® 90 concentration of 0.5% v/v. TABLE 4 Softanol ® 90 DoseDependence for Hydrolysis of PCS (5%) by Celluclast 1.5 L (2 mg/g PCS)at 50° C. Softanol ® 90, Softanol ® 90, 120-hour Improvement Reaction %ml/g PCS Conversion, % at 120 h, % Celluclast (2 mg/g 0.0 0.0 60 NA PCS)Celluclast (2 mg/g 0.25 0.05 72 21 PCS) + Softanol ® 90 0.5 0.1 78 301.0 0.2 72 20 1.5 0.3 67 13 2.0 0.4 67 13

Example 23 Enhanced Production of Sugars from PCS Using DifferentSoftanol® Products at 50° C.

Example 22 was repeated, except that different Softanol® products,Softanol® 50, 90, 120, or 200, with increasing degree of ethoxylationand increasing hydrophilic/lipophilic balance (HLB) values were added at0.2 ml per g of PCS (1.0% v/v) to reactions with 2 mg of Celluclast 1.5Lper g of PCS.

FIG. 22 shows that all Softanol® products performed similarly,increasing the final hydrolysis yield of reducing sugars by 17-30%compared to reactions without surfactants.

Example 24 Effect of Softanol® 90 on Hydrolysis of PCS by Celluclast1.5L and Trichoderma reesei Fermentation Broth Expressing Aspergillusoryzae Beta-Glucosidase at 50° C.

Example 22 was repeated, except that Softanol® 90 was added at 0.2 mlper g of PCS (1.0% v/v) to reactions with Celluclast 1.5L (2 mg/g PCS)supplemented with desalted and buffer exchanged Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae) at 0.06mg per g of PCS or to reactions with cell-free filtered fermentationbroth of Aspergillus oryzae beta-glucosidase (recombinantly produced inTrichoderma reesei) at 2 mg per g of PCS.

Without Softanol® 90, both enzymes performed similarly. FIG. 23 showsthat the addition of Softanol® 90 provided a comparable boosting effectfor both enzymes.

Example 25 Enhancing Effect of Softanol® 90 on Hydrolysis of PCS byTrichoderma reesei CBHI at 40-65° C.

Trichoderma reesei cellobiohydrolase I (CBHI) was isolated and purifiedto homogeneity from Celluclast 1.5L using methods described by Suurnakkiet al., 2000, Cellulose 7: 189-209.

Hydrolysis of ethanol-washed/milled PCS (1% w/v on a dry weight basis)by purified Trichoderma reesei CBHI (2, 5, and 10 mg/g PCS) was carriedout in 50 mM sodium acetate pH 5.0 in a 0.5-ml volume with intermittentstirring at 40° C., 50° C., 55° C., 60° C., and 65° C. Samples weretaken at different time-points and analyzed for reducing sugars asdescribed in Example 18. Reactions containing Trichoderma reesei CBHI at2 mg/g PCS were supplemented with 0.1% v/v Softanol® 90 (0.1 ml/g PCS),and the results were compared with control reactions run without thesurfactant.

The results are shown in FIG. 24. At 55° C., addition of Softanol® 90increased the 24-hour hydrolysis yield of reducing sugars by 94%compared to reaction without the surfactant (from 6.1% to 11.9%).Achieving a comparable hydrolysis yield in the absence of Softanol® 90required 2.5 times higher enzyme loading (5 mg/g PCS).

Example 26 Enhancing Effect of Softanol® 90 on Hydrolysis of PCS by(Trichoderma reesei CBHI plus Aspergillus fumigatus Beta-Glucosidase)Mixture at 40-65° C.

Example 25 was repeated, except that Trichoderma reesei CBHI wassupplemented with desalted and buffer exchanged Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae) at 5% ofthe CBHI protein loading (0.1, 0.25, and 0.5 mg/g PCS).

The results as shown in FIG. 25 demonstrated that at 55° C., addition ofSoftanol® 90 increased the 24-hour hydrolysis yield of reducing sugarsby 115% compared to reaction without the surfactant (from 9.8% to21.1%). Achieving comparable hydrolysis yield in the absence ofSoftanol® 90 required 2.5 times higher enzyme loading (5 mg/g PCS).

Example 27 Enhancing Effect of Softanol® 90 on Hydrolysis of PCS byTrichoderma reesei CBHI Plus Trichoderma reesei EGI Plus Aspergillusfumigatus Beta-Glucosidase Mixture at 40-65° C.

Example 25 was repeated, except that Trichoderma reesei CBHI (2, 5, 10,and 20 mg/g PCS) was supplemented with desalted and buffer exchangedTrichoderma reesei endoglucanase I (EGI; recombinantly produced inAspergillus oryzae) at 20% of the CBHI protein loading (0.4, 1, 2, and 4mg/g PCS) and desalted and buffer exchanged Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae) at 5% ofCBHI protein loading (0.1, 0.25, 0.5, and 1 mg/g PCS).

The results are shown in FIG. 26. At 55° C., addition of Softanol® 90increased the 24-hour hydrolysis yield of reducing sugars by 132%compared to reaction without the surfactant (from 11.9% to 27.6%). Theresulting hydrolysis yield was higher than that obtained using 2.5 timeshigher enzyme loading (5 mg/g PCS) in the absence of Softanol® 90 (27.6%vs. 21.3%).

Example 28 Enhancing Effect of Softanol® 90 on Hydrolysis of PCS byTrichoderma reesei CBHI Plus Acidothermus Cellulolyticus E1cd PlusAspergillus fumigatus Beta-Glucosidase Mixture at 40-65° C.

Example 27 was repeated, except that bacterial endoglucanase,Acidothermus cellulolyticus E1cd obtained from NREL, was used instead offungal endoglucanase, Trichoderma reesei EGI. Acidothermuscellulolyticus E1cd was expressed from Streptomyces lividans TK24acccording to U.S. Pat. No. 5,275,944. A truncated form of this enzymewas produced by subjecting the Streptomyces lividans-expressed enzyme toproteolytic treatment to remove the cellulose-binding domain (Baker etal., 1995, in Enzymatic Degradation of Insoluble Polysaccharides,Saddler, J. N. and Penner, M. H., eds., ACS Series 618, AmericanChemical Society, Washington, D.C., 113-141).

The results as shown in FIG. 27 demonstrated that at 55° C., addition ofSoftanol® 90 increased the 24-hour hydrolysis yield of reducing sugarsby 70% compared to reaction without the surfactant (from 22.2% to37.7%). The resulting hydrolysis yield was higher than that obtainedusing 2.5 times higher enzyme loading (5 mg/g PCS) in the absence ofSoftanol® 90 (37.7% vs. 33%).

Example 29 Effect of Softanol® 90 on Hydrolysis of Avicel by Celluclast1.5L at 50° C.

Hydrolysis of microcrystalline cellulose, Avicel PH101 (1% w/v on a dryweight basis, FMC Corporation, Philadelphia, Pa.) by Celluclast 1.5L(1.25, 2.5, 5, 10, and 20 mg/g cellulose) supplemented with desalted andbuffer exchanged Aspergillus oryzae beta-glucosidase (recombinantlyproduced in Aspergillus oryzae) at 0.6 mg per g of cellulose was carriedout in 50 mM sodium acetate pH 5.0 in a 1-ml volume with intermittentstirring at 50° C. Samples were taken at different time-points andanalyzed for reducing sugars as described in Example 18. Reactionscontaining Celluclast 1.5L at 1.25, 2.5 and 5 mg/g cellulose weresupplemented with Softanol® 90 at 0.05, 0.1 and 0.2 ml/g cellulose, andthe results were compared with control reactions without the surfactant.

FIG. 28 shows that Softanol® 90 had no significant boosting effect onhydrolysis of Avicel under the above conditions.

Example 30 Enhanced Production of Sugars from PCS Using Non-IonicSurfactants at 50° C.

Hydrolysis of water-washed/milled PCS (5% w/v on a dry weight basis) bycell-free filtered fermentation broth of Aspergillus oryzaebeta-glucosidase (recombinantly produced in Trichoderma reesei) at 2 mgper g of PCS was carried out in the presence of various non-ionicsurfactants (0.1 ml/g PCS) as shown in Table 5 in a 2-ml volume withintermittent stirring at pH 5.0, 50° C. for 71 hours. Samples were takenat different time-points and analyzed for reducing sugars as describedin Example 18. The results were compared to a control reactioncontaining no surfactant. TABLE 5 Effect of Non-Ionic Surfactants (0.1ml/g PCS) on Hydrolysis of PCS (5%) by Trichoderma reesei FermentationBroth with Expressed Aspergillus oryzae Beta-Glucosidase (2 mg/g PCS) at50° C. Improvement at 71 Surfactant 71-hour Conversion, % hours, % None60 NA Softanol ® 50 66 8.7 Softanol ® 90 72 18.8 Softanol ® 120 69 14.8Softanol ® 200 71 18.3 Lutensol ® AT50 71 18.3 Lutensol ® AT80 69 13.8Tergitol ™ NP-9 66 10.2 Novell ™ II TDA-6.6 70 16.5 Novell ™ II TDA-8.569 14.4 Brij ™ 35 70 16.1 Brij ™ 56 70 16.5 Brij ™ 97 63 3.9 Brij ™ 9871 18.4 Pluronic ® F-68 71 18.1

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:Deposit Accession Number Date of Deposit E. coli TOP10 (pEJG113) NRRLB-30695 Oct. 17, 2003

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method for degrading a lignocellulosic material, comprising:treating the lignocellulosic material with an effective amount of one ormore cellulolytic enzymes in the presence of at least one surfactantselected from the group consisting of a secondary alcohol ethoxylate,fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate,and polyoxyethylene ether, wherein the presence of the surfactantincreases the degradation of lignocellulosic material compared to theabsence of the surfactant.
 2. The method of claim 1, wherein thelignocellulosic material is selected from the group consisting ofherbaceous material, agricultural residue, forestry residue, municipalsolid waste, waste paper, and pulp and paper mill residue.
 3. The methodof claim 1, wherein the lignocellulosic material is corn stover.
 4. Themethod of claim 1, wherein the one or more cellulolytic enzymes areselected from the group consisting of a cellulase, endoglucanase,cellobiohydrolase, and beta-glucosidase.
 5. (canceled)
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 19. The method of claim 1, furthercomprising treating the lignocellulosic material with an effectiveamount of one or more enzymes selected from the group consisting of ahemicellulase, esterase, protease, laccase, peroxidase, or a mixturethereof.
 20. The method of claim 19, wherein the esterase is a lipase,phospholipase, cutinase, or a mixture thereof.
 21. The method of claim1, wherein the method is a pretreatment process.
 22. The method of claim1, wherein the method is a step in a simultaneous saccharification andfermentation process (SSF).
 23. The method of claim 1, wherein themethod is a step in a hybrid hydrolysis and fermentation process (HHF).24. The method of claim 1, further comprising recovering the degradedlignocellulosic material.
 25. The method of claim 24, wherein thedegraded lignocellulosic material is a sugar.
 26. The method of claim24, wherein the sugar is selected from the group consisting of glucose,xylose, mannose, galactose, and arabinose.
 27. A method for producing anorganic substance, comprising: (a) saccharifying a lignocellulosicmaterial with an effective amount of one or more cellulolytic enzymes inthe presence of at least one surfactant selected from the groupconsisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate,nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether,wherein the presence of the surfactant increases the degradation oflignocellulosic material compared to the absence of the surfactant; (b)fermenting the saccharified lignocellulosic material of step (a) withone or more fermentating microoganisms; and (c) recovering the organicsubstance from the fermentation.
 28. The method of claim 27, wherein thelignocellulosic material is selected from the group consisting ofconsisting of herbaceous material, agricultural residue, forestryresidue, municipal solid waste, waste paper, and pulp and paper millresidue.
 29. The method of claim 27, wherein the lignocellulosicmaterial is corn stover.
 30. The method of claim 27, wherein the one ormore cellulolytic enzymes are selected from the group consisting of acellulase, endoglucanase, cellobiohydrolase, and beta-glucosidase. 31.(canceled)
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 44. (canceled)45. The method of claim 27, further comprising treating thelignocellulosic material with an effective amount of one or more enzymesselected from the group consisting of a hemicellulase, esterase,protease, laccase, peroxidase, or a mixture thereof.
 46. The method ofclaim 45, wherein the esterase is a lipase, phospholipase, cutinase, ora mixture thereof.
 47. The method of claim 27, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.
 48. The method of claim 27, wherein the organic substanceis an alcohol, organic acid, ketone, amino acid, or gas.
 49. (canceled)50. (canceled)
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